Immunoglobulin variable domains

ABSTRACT

VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.16/695,420, filed Nov. 26, 2019, which is a divisional application ofU.S. application Ser. No. 15/311,564, filed Nov. 16, 2016, which is anational stage filing under 35 U.S.C. § 371 of International ApplicationNo. PCT/EP2015/060643, filed May 13, 2015, which was published under PCTArticle 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 61/994,552, filed May16, 2014, U.S. provisional application Ser. No. 62/014,015, filed Jun.18, 2014, U.S. provisional application Ser. No. 62/040,167, filed Aug.21, 2014, U.S. provisional application Ser. No. 62/047,560, filed Sep.8, 2014, and U.S. provisional application Ser. No. 62/133,600, filedMar. 16, 2015, the entire contents of each of which is incorporated byreference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 9, 2020, isnamed A084870168US07-SEQ-CRP, and is 1,309,001 bytes in size.

DESCRIPTION

The present invention relates to improved heavy-chain immunoglobulinvariable domains.

The invention in particular refers to improved heavy-chainimmunoglobulin variable domains that either have an exposed C-terminalregion or end (as further described herein; see also WO 12/175741) orthat are used in (or intended for use in) applications where they havean exposed C-terminal region or end (again, as further describedherein). Some preferred but non-limiting examples of the former areimmunoglobulin single variable domains (also referred to herein as“ISV's” or “ISVD's”) such as Nanobodies (including VHH's, humanizedVHH's and camelized VH's such as camelized human VH's), (single domain)antibodies that are VH domains or derived from VH domains, and dAb'sthat are VH domains or derived from VH domains. Some preferred butnon-limiting examples of the latter are VH domains that are used in (orintended for use in) single chain FV's (ScFv's) or diabodies.

The invention also relates to proteins, polypeptides and otherconstructs, molecules or chemical entities that comprise or essentiallyconsist of (one or more of) the improved heavy-chain immunoglobulinvariable domains of the invention as described herein; to methods forexpressing/producing the improved heavy-chain immunoglobulin variabledomains of the invention and/or for expressing/producing proteins,polypeptides and other constructs, molecules or chemical entitiescomprising the same; to compositions and products (such aspharmaceutical compositions and products) that comprise the improvedheavy-chain immunoglobulin variable domains of the invention and/orproteins, polypeptides and other constructs, molecules or chemicalentities comprising the same; to nucleotide sequence and nucleic acidsthat encode the improved heavy-chain immunoglobulin variable domains ofthe invention and/or that encode proteins or polypeptides comprising thesame; and to uses (and in particular therapeutic, prophylactic anddiagnostic uses) of the improved heavy-chain immunoglobulin variabledomains of the invention and of proteins, polypeptides and otherconstructs, molecules or chemical entities comprising the same.

Further aspects, embodiments, advantages, applications and uses of theinvention will become clear from the further description herein.

In the present application, the amino acid residues/positions in animmunoglobulin heavy-chain variable domain will be indicated with thenumbering according to Kabat. For the sake of convenience, FIG. 1 givesa table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT. Note: for thepresent description and claims, Kabat numbering is decisive; othernumbering systems are given for reference only).

Also, in the invention, an immunoglobulin variable domain is said tohave “an exposed C-terminal end or region” when it is not associatedwith or linked to a constant domain (such as a C_(H)1 domain). Referenceis made to the relevant prior art cited herein.

In particular, as described in WO 12/175741, the C-terminal region (asthis term is also used herein) is part of a putative epitope on the ISVthat also includes, among other residues, the amino acid residue atposition 14 (and the amino acid residues next/close to the same in theamino acid sequence, such as positions 11, 13 and 15) and may alsocomprise the amino acid residue at position 83 (and the amino acidresidues next/close to the same in the amino acid sequence, such aspositions 82, 82a, 82b and 84) and/or the amino acid residue at position108 (and the amino acid residues next/close to the same in the aminoacid sequence, such as positions 107. As in WO 12/17574, this putativeepitope is also collectively referred to herein as the “C-terminalregion”, it being understood that this C-terminal region at leastcomprises the C terminal sequence VTVSS (i.e. each of positions 109,110, 111, 112 and 113) and the amino acid residue at position 14, andmay also comprise the amino acid residues at positions 83 and 108, andpossibly also the amino acid residues at positions 13, 15, 82b, 83, 84and 107.

As a result of research into single chain Fv's or “ScFv's” (which areconstructs that contain immunoglobulin variable domains that, similar toISVD's, are not associated with constant domains), it has been describedin the art that the C-terminus of an immunoglobulin variable domaincontains a hydrophobic patch that in a conventional full-sized antibodyis buried in the interface between the variable domain and the constantdomain but that becomes solvent-exposed when the variable domain is notassociated with a constant domain (see for example Nieba et al., ProteinEngineering, 10, 435-444 (1997) and Harmsen et al., Molecular Immunology(2000), 579-590).

It is also well known that epitopes that are usually buried within thestructure of a protein (also referred to as “neo-epitopes” or “crypticepitopes”) may trigger the immune system once they becomesolvent-exposed, for example due to degradation, misfolding oraggregation of the protein involved. For example, in the case of buriedhydrophobic portions of biomolecules (so-called “hyppos”), it has beensuggested that these form part of a general damage-associated molecularpattern that leads to innate immune responses once the hyppos becomesolvent-exposed (see for example Seong and Matzinger, Nature Reviews2004, 469), and various examples of previously-buried hydrophobicpatches triggering immune responses have been described in the art (seefor example David et al., JBC, 2001, 6370-6377; Matsuura et al.,International Immunology, 2000, 1183-1192; Rasheed et al., Life Sciences79 (2000), 2320-2328). More generally, it is also known in the art thathydrophobic amino acids are prone to be part of B-cell epitopes (see forexample WO 11/07586, page 10; and Kolaskar, FEBS 276, 172-174 (1990)).Similarly, it has been described that the hydrophobic patch at theC-terminus of a heavy-chain variable domain (as described by Nieba etal. and Harmsen et al., supra) may form B-cell epitopes which can giverise to and/or interact with (emerging and/or pre-existing) anti-drugantibodies (WO 11/07586). For this reason, it has been proposed to makemutations to some of the amino acid residues that form part of theC-terminus of the variable domains to reduce hydrophobicity and/or toremove B-cell epitopes. For example, Nieba et al. suggest to mutatepositions 11, 14, 41, 84, 87 and/or 89 of a VH region (numberingaccording to Kabat), whereas in WO 11/07586 it is suggested to mutatepositions 99, 101 and/or 148 (AHo numbering) of a VL domain or positions12, 97, 98, 99, 103 and/or 144 of a VH domain (again AHo numbering—thesepositions correspond to positions 11, 83, 84, 85, 89 and 103 accordingto Kabat). Similarly, Harmsen et al. suggest to mutate positions 12 and101 (IMGT numbering; these are positions 11 and 89 according to Kabat)to compensate for the absence of a C_(H)1 domain; and they also identifya specific subfamily of VHH's (called “VHH4's”) that contain amino acidsthat are suitable candidates for substitutions at these positions.

It has also been described in the art (see for example WO 12/175741 andthe references cited in the next paragraphs) that biological samplesobtained from human subjects may contain (pre-existing) proteins orfactors that are capable of binding to the exposed C-terminal region orend of an immunoglobulin variable domain (for example, the C-terminalregion or end of an ISVD or of a VH or VL domain in an ScFv or diabody).

For example, WO 2013/024059 states that “in sera from some healthy naivehuman subjects, pre-existing anti-VH autoantibodies are present that canbind both VH domain antibodies and VHH molecules, as well as anti-VL (egV kappa (VK)) autoantibodies that can bind VL molecules.”, and that “thepre-existing ADAs that bind VH dAbs are similar to anti-hinge antibodiesin that they bind IgG fragments but not those same sequences found insitu on intact IgG.”

Holland et al., J. Clin. Immunol. 2013, 33(7):1192-203 describe that theblood of around half of normal healthy humans contain varying levels ofa new class of anti-IgG autoantibodies that can bind to the frameworksequences of fully human V_(H) domain antibodies (which Holland et al.also refer to as “HAVH auto-antibodies”). Holland et al. further mentionthat these auto-antibodies appear to be predominantly of the IgGisotype, display a relatively high affinity (about 10⁻¹⁰M) affinity forV_(H) sequences, and that a free C-terminus appears to be important forthe binding of these HAVH autoantibodies to V_(H) domains.

The issues relating to pre-existing biotherapeutic-reactive antibodiesagainst biotherapeutic molecules and their regulatory impact are alsogenerally discussed by Xue et al., AAPS J. 2013; 15(3):852-5.

The aforementioned prior art has also focused on ways in which thesequence of an immunoglobulin variable domain may be modified so as toprevent or reduce binding of such pre-existing antibodies/factor(s) tothe variable domains. In this respect, WO 2011/07586 suggests to makeone or more mutations in the amino acid sequence of the variable domainat some specific positions of the domain (which positions aresurface-exposed). WO 12/175741 describes that the binding of suchpre-existing antibodies/factors may be reduced by adding a few aminoacid residues (and as little as one alanine residue) to the C-terminalend of the VH-domain and/or by making one or more specific substitutionsor deletions within the C-terminal region of the variable domain, whichis described in WO 12/175741 as at least comprising the C-terminal aminoacid sequence VTVSS and the amino acid residue at position 14 (for whichposition WO 12/175741 teaches that the presence of an alanine residueprovides for reduced binding of pre-existing antibodies as compared tothe presence of the “human” amino acid residue proline), and possiblyalso the amino acid residues at positions 108 and 83 and amino acidresidues close to said positions (WO 2013/024059 provides essentiallythe same teaching as WO 12/175741).

For example, in research performed by applicant/assignee leading up tothe filing of WO 12/175741, it has been found that adding a singlealanine residue to the C-terminal region or end of an exposed VH domainwill usually prevent/remove (essentially all of) the binding ofpre-existing antibodies/factors that are present in samples obtainedfrom most human subjects (see for example page 62, lines 20-25 and page57, line 30 to page 58, lines 3 of WO 12/175741); and these findingshave been confirmed by additional results that were obtained byapplicant/assignee after the filing of WO 12/175741 when the C-terminalalanine substitution of WO 12/175741 was applied to other Nanobodies(data not shown).

Also, in WO 12/175741 as well as in WO 12/175400 by applicant/assignee,the C-terminal extensions described in WO 12/175741 are applied tocertain serum-albumin-binding Nanobodies (see for example WO 12/175741:SEQ ID NO's: 37, 51-53 and 55-64 and the constructs shown in SEQ IDNO's: 41, 43 and 44; and WO 12/175400: SEQ ID NO's: 6 to 11).

FIG. 9 of WO 12/175741 also describes two albumin-binding sequences thatare used as reference sequences in the Experimental Part below. Theseare SEQ ID NO:37 from FIG. 9 of WO 12/175741 (also referred to herein as“Reference B”; its sequence is given herein as SEQ ID NO: 45) and “SEQID NO:37 without the added C-terminal amino acid residues” from FIG. 9of WO 12/175741 (also referred to herein as “Reference A”; its sequenceis given in SEQ ID NO:44). Reference A and Reference B are both derivedfrom the sequence of the humanized anti-albumin Nanobody “Alb-8” that isgiven as SEQ ID NO:62 in WO 06/122787 (and that is also referred toherein as “Alb-11”); but, compared to the sequence of Alb-11, ReferenceA comprises an N-terminal His tag; and Reference B comprises anN-terminal His tag and a C-terminal alanine residue. Reference A,Reference B and Alb-8/Alb-11 all contain the CDRs given in SEQ ID NO's:41 to 43, respectively.

Other examples of Nanobodies and other immunoglobulin single variabledomains that have C-terminal extensions and/or mutations in theC-terminal region can for example be found in the following prior art:WO 06/129843 (see for example SEQ ID NO's: 4, 6, 8 and 10); WO 03/035695(see for example some of the sequences listed on pages 61-64); Vu etal., Molecular Immunology, 1121-1131, 1997 (see for example some of thesequences listed in FIG. 2); WO 11/003622 (see for example the sequencesgiven as SEQ ID NO's: 10 to 27); WO09/058383 (see for example thesequence TAR2h-10-27 mentioned on page 51); WO 10/042815 (see forexample the sequences of SEQ ID NO's: 15, 17, 27 and 30); and WO04/044204 (see for example the sequences of SEQ ID NO's: 31, 35, 37, 47and 49).

Some of the references cited herein also give examples of ISVD sequencesin which the last C-terminal amino acid of the ISVD is an amino acidother than serine (S), for example because the serine at position 113has been replaced by another amino acid and/or because the serine atposition 113 has been deleted and a C-terminal amino acid has been added(in practice, the end-result in terms of the C-terminal amino acidsequence will be the same).

Some of the references cited herein also give examples of Nanobodies andother immunoglobulin single variable domains in which position 112 is anamino acid other than serine. For example, WO 12/175741 describesNanobodies in which position 112 is glycine (G); Vu at al. (supra)describes Nanobodies in which position 112 is alanine (A) or isoleucine(I); WO 13/024059 exemplifies a S112A substitution; and WO 08/020079cited below exemplifies an S112F substitution and also generally statesthat the Nanobodies described therein can contain a limited number ofamino acid residues added at the carboxy-terminal end of the amino acidsequence of the Nanobody.

In the research leading up to the present invention, after havingestablished that adding C-terminal extension (which may be as simple asa single C-terminal alanine residue, see again WO 12/175741, Example 3)to the C-terminal region or end of a nanobody essentiallyprevents/removes binding of pre-existing antibodies/factors in mostsamples of human subjects/patients, it was investigated whether samplesobtained from human subjects (healthy volunteers and/or subjectssuffering from a disease or disorder) possibly contain (other)pre-existing antibodies or factors that can bind to the exposedC-terminal region of a nanobody (or other VH domain) even when aC-terminal extension is present. In doing so, the present inventors havefound that, although essentially no such pre-existing antibodies bindingto a C-terminally extended VH domain can be found in the blood or serumof healthy volunteers or in blood or serum obtained from human patientssuffering from one of a number of different diseases (including someinflammatory diseases or auto-immune disorders—data not shown), someblood or serum samples that have been obtained from certain (but notall) human subjects suffering from certain severe (auto-)immunedisorders (such as systemic lupus erythematosus; also abbreviated hereinas “SLE”) appear to contain some pre-existing antibodies/factors thatcan bind to nanobodies even when said nanobodies comprise a C-terminalextension.

Thus, generally, the purpose of the present invention is to provideimproved heavy-chain immunoglobulin variable domains (and in particularimproved heavy-chain ISVD's and more in particular improved nanobodies)that, when they have an exposed C-terminal region or end, are less proneto be bound by pre-existing antibodies/factors, such as those found inblood or serum samples obtained from human subjects.

In particular, the purpose of the present invention is to provideimproved heavy-chain immunoglobulin variable domains that, when theyhave an exposed C-terminal region or end, are less prone to binding bypre-existing antibodies/factors (again, such as those found in blood orserum samples obtained from human subjects) that can still bind to theexposed C-terminal region or end of the heavy-chain variable domain whensaid domain comprises a C-terminal extension (for example, as describedin WO 12/175741, WO 13/024059 and the further prior art cited herein).

As mentioned herein, such pre-existing antibodies that can bind to aheavy-chain variable domain with a C-terminal extension have been found,by the present inventors, to be present in blood or serum samplesobtained from human subjects suffering from certain (auto-) immunediseases or disorders that severely impact/activate the immune system(such as SLE).

Thus, more in particular, the purpose of the present invention is toprovide improved heavy-chain immunoglobulin variable domains (and inparticular improved heavy-chain ISVD's and more in particular improvednanobodies) that, when they have an exposed C-terminal region or end,are less prone to binding by pre-existing antibodies/factors, such asthose that are found in blood or serum samples obtained from humansubjects suffering from certain (auto-) immune diseases or disordersthat severely impact/activate the immune system (such as SLE).

Even more in particular, the purpose of the present invention is toprovide improved heavy-chain immunoglobulin variable domains (and inparticular improved heavy-chain ISVD's and more in particular improvednanobodies) that, when they have an exposed C-terminal region or end,are less prone to binding by those pre-existing antibodies/factors thatare found in blood or serum samples obtained from human subjectssuffering from certain (auto-) immune diseases or disorders and that canstill bind to the exposed C-terminal region or end of the VH domain whenthe VH domain comprises a C-terminal extension.

It has now been found that the binding of pre-existingantibodies/factors to a heavy-chain variable domain with an exposedC-terminal end can be (further) reduced by a mutation of the serine atposition 112 (Kabat numbering) to either lysine (K) or glutamine (Q). Inparticular, it has been found that such an S112K or S112Q mutation can(further) reduce or essentially prevent/remove binding of pre-existingantibodies/factors that can bind to a heavy-chain variable domain thatcomprises a C-terminal extension (but no S112K or S112Q mutation), suchas those pre-existing antibodies/factors that are found in the blood orserum of human subjects suffering from severe auto-immune disorders suchas SLE.

It has also been found that introducing the specific mutations disclosedherein (and in particular the following mutations L11V in combinationwith V89L, and optionally further in combination with T110K) mayimprove, or contribute to a (further) improvement of, the solubility ofimmunoglobulin single variable domains, such as the ISVD's generally andspecifically disclosed herein (data not shown).

Thus, in a first aspect, the invention relates to an immunoglobulinheavy-chain variable domain (VH domain) in which the amino acid residueat position 112 (Kabat numbering) is either a lysine (K) residue or aglutamine (Q) residue. Such an immunoglobulin heavy-chain variabledomain is also referred to herein as a “VH domain of the invention”.When a VH domain of the invention is an immunoglobulin single variabledomain (as is preferred), it will also be referred to herein as an “ISVDof the invention”. Similarly, when the VH domain of the invention is aNanobody (as is even more preferred), it will also be referred to hereinas a “Nanobody of the invention”.

Generally, the VH domains of the invention either have an exposedC-terminal end or region, and/or are present in a protein, polypeptide,compound, entity or construct in which they have an exposed C-terminalend or region, and/or are intended for a use in which they have anexposed C-terminal region (for example, for use in in a protein,polypeptide, compound, entity or construct in which they are intended toform the C-terminal end or region).

In one aspect of the invention, the VH domain of the invention is an(heavy-chain) immunoglobulin single variable domain, meaning aheavy-chain variable domain that can form a functional antigen bindingsite without interaction with a VL domain. For example, the VH domain ofthe invention can be a Nanobody (including a VHH, a humanized VHH and/ora camelized VHs such as camelized human VH's), a (single) domainantibody that is a VH domain or that is derived from a VH domain, or adAb that is a VH domain or that is derived from a VH domain. The VHdomain of the invention is preferably a nanobody (e.g. a VHH domain, ahumanized VHH domain or a camelized VH domain such as a camelized humanVH domain).

According to another aspect of the invention, the VH domain of theinvention can be a heavy-chain variable domain that, in the protein,polypeptide, protein or construct in which is it present, does requirean interaction with a VL domain in order to form an antigen binding siteand that does have or form an exposed C-terminal end or region. Forexample, a VH domain according to this aspect of the invention can be aVH domain that is present in and/or used in an ScFv and or a diabody.

According to a more specific aspect of the invention, the VH domain ofthe invention has a C-terminal sequence (positions 109 to 113 accordingto Kabat) that is VTVKS (SEQ ID NO:1) or VTVQS (SEQ ID NO:2), or asequence that has one amino acid difference (i.e. at one of thepositions 109, 110, 111 or 113) with either the sequence VTVKS and/orVTVQS and that still has either a lysine (K) or glutamine (Q) atposition 112.

Also, as further described herein, in one particularly preferred aspectof the invention, the VH domain of the invention also contains aC-terminal extension (e.g. as described in WO 12/175741 and/or in WO13/024059) at its C-terminal end (i.e. linked to the serine residue atthe end of the VTVKS-, VTVQS- or similar motif), in particular aC-terminal extension that is as further defined herein. However, as alsofurther described herein, it is also possible that the VTVKS-, VTVQS- orsimilar motif forms the C-terminal end of the VH domain (although thiswill usually be less preferred) or that the VH domain of the inventionis linked at its C-terminal end (optionally via a suitable linker) toanother amino acid sequence, moiety, domain or binding unit. Forexample, when the VH domain of the invention is an ISVD, the VH domainmay be linked at its C-terminal end to another ISVD, optionally via alinker (and said other ISVD may then also be a VH domain of theinvention).

Overall, as is well known for immunoglobulin variable domains generally,the VH domains of the invention will comprise 4 framework regions (FW1,FW2, FW3 and FW4) and 3 CDR's (CDR1, CDR2 and CDR3). As withimmunoglobulin variable domains generally, the sequence of the CDR'swill depend on the antigen/target(s) to which the VH domains of theinvention have been raised and/or are intended to bind. The frameworkregions can generally be any suitable framework regions for VH domains(albeit that position 112 and/or position 89 will be as furtherdescribed herein). If the VH domain of the invention is an ISVD, the VHdomain will have framework sequences that are suitable for an ISVD(optionally in association with one or more of the CDR's). For example,if the VH domain of the invention is a Nanobody, the framework regionswill generally contain a suitable number of VHH hallmark residues (forwhich reference is for example made to WO 08/020079 and to some of theother patent applications of applicant/assignee cited herein).

Thus, for example, when the VH domains of the invention are Nanobodies,said Nanobodies of the invention can contain one or more of the“Hallmark residues” that are characteristic of VHH's/Nanobodies (e.g. atpositions 11, 37, 44, 45, 47, 83, 84, 103, 104 and/or 108; see forexample Tables A-3 and A-5 to A-8 of WO 08/020079); one or more otheramino acid residues that can be present in VHH's/Nanobodies (such as oneor more humanizing substitutions that are known per se for VHH's andNanobodies; reference is for example made to the teaching in WO08/020079; see again the previously mentioned Tables A-3 and A-5 to A-8)and/or one or more other suitable amino acid residues or substitutionsfor VHH's/Nanobodies; or any suitable combination of such amino acidresidues/substitutions.

Nanobodies of the invention in which position 112 is K or Q (i.e. withor without a C-terminal extension) preferably contain an amino acid atposition 11 that is chosen from L (the most often occurring amino acidresidue in VHH's), E, K, M, S, V, W or Y; more preferably from L, E, K,V or Y, and even more preferably from L, K or V (with V being mostpreferred). For example and without limitation, compared to the leucineresidue that most often occurs in VHH's, they can contain an L11K orL11V mutation. They can also, but without limitation, for examplecontain a Q108L mutation (a well-known humanizing substitution forVHH's/Nanobodies). Other amino acid residues that can be present (again,without limitation, and for example other amino acid residues thatnaturally occur at this position in human VH's or VHH's may also bepresent at these positions) are for example one or more of an alanine(A) at position 14 (which is a very frequently occurring amino acidresidue at this position in naturally occurring VHH's), a proline atposition 14 (which is the most common amino acid at this position inhuman VH domains), as well as the mutations suggested by Harmsen et al.(in particular, those that Harmsen et al suggest based on the sequenceof VHH4-class VHH's, such as V89M or V89T) and/or (other) mutations atthe positions suggested by Nieba (for example, at one or more ofpositions 11, 87 and/or 89, see Nieba, page 437, right hand column).Another suitable mutation is for example T110K or T110Q. Also: (i)position 41 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 41 and may in particular be (or be chosenfrom) proline (P), serine (S), threonine (T), alanine (A) or leucine(L), which are some of the amino acid residues that most frequentlyoccur at this position in either humans or llamas, and may more inparticular be either proline (P) or alanine (A); and/or (ii) Also,position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A)

According to a more specific aspect of the invention, a VH domain of theinvention (which is as further described herein) has a framework 4 (FW4sequence) that is either:

-   a) is one of the FW4 sequences of SEQ ID NO's: 3 to 20 mentioned in    Table 1 below

TABLE 1 FW4 sequences WGQGTQVTVKS (SEQ ID NO: 3)WGKGTLVTVKS (SEQ ID NO: 4) RGQGTRVTVKS (SEQ ID NO: 5)WGLGTQVTISS (SEQ ID NO: 6) GSQGTQVTVKS (SEQ ID NO: 7)LRGGTQVTVKS (SEQ ID NO: 8) RGQGTLVTVKS (SEQ ID NO: 9)RSRGIQVTVKS (SEQ ID NO: 10) WGKGTQVTVKS (SEQ ID NO: 11)WGQGTQVTVQS (SEQ ID NO: 12) WGKGTLVTVQS (SEQ ID NO: 13)RGQGTRVTVQS (SEQ ID NO: 14) WGLGTQVTISS (SEQ ID NO: 15)GSQGTQVTVQS (SEQ ID NO: 16) LRGGTQVTVQS (SEQ ID NO: 17)RGQGTLVTVQS (SEQ ID NO: 18) RSRGIQVTVQS (SEQ ID NO: 19)WGKGTQVTVQS (SEQ ID NO: 20)or:

-   b) is a sequence that has no more than three, preferably no more    than two amino acid differences with at least one of the FW4    sequences of SEQ ID NO's 3 to 20, in which (i) the amino acid    residue at the position corresponding to position 112 of the Kabat    numbering is either K or Q; and in which (ii) the amino acid residue    at the position corresponding to position 103 of the Kabat numbering    is preferably W or R; (iii) the amino acid residue at the position    corresponding to position 104 of the Kabat numbering is preferably    G; (iv) the amino acid residue at the position corresponding to    position 106 of the Kabat numbering is preferably G; (v) the amino    acid residue at the position corresponding to position 107 of the    Kabat numbering is preferably T; (vi) the amino acid residue at the    position corresponding to position 108 of the Kabat numbering is    preferably Q or L (and in humanized nanobodies preferably L); (vii)    the amino acid residue at the position corresponding to position 109    of the Kabat numbering is preferably V; (viii) the amino acid    residue at the position corresponding to position 110 of the Kabat    numbering is preferably T (or alternatively may be K or Q); (ix) the    amino acid residue at the position corresponding to position 111 of    the Kabat numbering is preferably V. Table 2 below gives some    non-limiting examples of amino acid residues that can be present at    the different positions (numbered according to Kabat) of such FW4    sequences.

TABLE 2 Examples of amino acid residues that can be present in the FW4sequences of the VH domains of the invention. Pos. Amino acidresidue(s): 103 W, R, G, S, K, A, M, Y, L, F, T, N, V, Q, P, E, C;preferably W 104 G, A, S, T, D, P, N, E, C, L; preferably G 105 Q, K, H,R, P, E, L, T, N, S, V, A, M, G 106 G, R, E 107 T, Q, I, A, S, N, R, V,D 108 Q, L, R, P, E, K, S, T, M, A, H; preferably Q or L 109 V, I, L 110T, S, N, A, I, F, K, Q 111 V, I, A 112 K or Q (invention) 113 S, T, A,L, P, F, E, V

Preferably, the VH domain of the invention has a framework 4 (FW4sequence) that is either:

a) (SEQ ID NO: 3) WGQGTQVTVKS or (SEQ ID NO: 12) WGQGTQVTVQS;or

-   b) a sequence that has no more than three, preferably no more than    two amino acid differences (such as only one amino acid difference)    with SEQ ID NO:3 and/or SEQ ID NO: 12, in which (i) the amino acid    residue at the position corresponding to position 112 of the Kabat    numbering is either K or Q; and in which (ii) the amino acid residue    at the position corresponding to position 103 of the Kabat numbering    is preferably W or R; (iii) the amino acid residue at the position    corresponding to position 104 of the Kabat numbering is preferably    G; (iv) the amino acid residue at the position corresponding to    position 106 of the Kabat numbering is preferably G; (v) the amino    acid residue at the position corresponding to position 107 of the    Kabat numbering is preferably T; (vi) the amino acid residue at the    position corresponding to position 108 of the Kabat numbering is    preferably Q or L (and in humanized nanobodies preferably L); (vii)    the amino acid residue at the position corresponding to position 109    of the Kabat numbering is preferably V; (viii) the amino acid    residue at the position corresponding to position 110 of the Kabat    numbering is preferably T (or alternatively may be K or Q); (ix) the    amino acid residue at the position corresponding to position 111 of    the Kabat numbering is preferably V. Again, Table 2 gives some    non-limiting examples of amino acid residues that can be present at    the different positions (numbered according to Kabat) of such FW4    sequences.

As described further herein, according to a preferred aspect of theinvention, VH domains of the invention that comprise a FW4 sequence asdescribed above preferably also contains a C-terminal extension (asfurther described herein). However, as also further described herein, itis also possible that the FW4 sequence forms the C-terminal end of theVH domain (although this will usually be less preferred) or that the VHdomain of the invention is linked at its C-terminal end (optionally viaa suitable linker) to another amino acid sequence, moiety, domain orbinding unit. For example, when the VH domain of the invention is anISVD, the VH domain may be linked at its C-terminal end to another ISVD,optionally via a linker (and said other ISVD may then also be a VHdomain of the invention).

As indicated herein, according to a preferred but non-limiting aspect ofthe invention, the VH domains of the invention contain a C-terminalextension, such as a C-terminal extension that is as described in WO12/175741 and/or in WO 13/024059, and in particular as described in WO12/175741

Thus, according to this aspect, the VH domain of the invention is animmunoglobulin heavy-chain variable domain (VH domain) in which: (i) theamino acid residue at position 112 (Kabat numbering) is not a serineresidue, and is preferably either a lysine (K) residue or a glutamine(Q) residue; and that (ii) at its C-terminal end (i.e. linked to theamino acid residue at position that is or corresponds to position 113according to the Kabat numbering) is linked to a further amino acidsequence (i.e. the “C-terminal extension”) that comprises between 1 and5 (such as 1, 2, 3, 4 or 5, and preferably 1, 2 or 3, and mostpreferably only 1 or 2 such as only 1) amino acid residues that are eachindependently chosen from suitable amino acid residues, and preferablyeach independently chosen from naturally occurring amino acids, and morepreferably each independently chosen from the group consisting ofalanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)(however, as can be seen from the data presented in WO 12/17574, otheramino acid residues such as serine, proline, threonine and/or lysine canalso be used as part of the C-terminal extension).

In particular, according to this aspect of the invention, the VH domainof the invention preferably has a C-terminal sequence that isVTVKS(X)_(n) (SEQ ID NO:21) or VTVQS(X)_(n) (SEQ ID NO:22) (or that isan amino acid sequence that, at the positions of the VTVKS motif or theVTVQS-motif, has one amino acid difference with either the sequenceVTVKS and/or VTVQS and that still has either a lysine (K) or glutamine(Q) at position 112), in which (i) the amino acid residues of the VTVKS-or VTVQS-motif (or VTVKS- or VTVQS-like motif) correspond to positions109 to 113 of the VH domain according to the Kabat numbering; (ii) n is1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or2, such as 1); and (iii) each X is an (preferably naturally occurring)amino acid residue that is independently chosen, and preferablyindependently chosen from the group consisting of alanine (A), glycine(G), valine (V), leucine (L) or isoleucine (I).

More in particular, according to this aspect of the invention, the VHdomain of the invention may have, as its FW4 sequence, one of the FW4sequences of SEQ ID NO's 3 to 20 (or an amino sequence that has no morethan three, preferably no more than two amino acid differences with atleast one of the FW4 sequences of SEQ ID NO's 3 to 20, in which theamino acid residue at the position corresponding to position 112 of theKabat numbering is either K or Q), in which said FW 4 sequence islinked, at its C-terminal end, to a C-terminal extension (X)_(n), inwhich n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I).

Accordingly, in this aspect of the invention, a VH domain of theinvention may have, at its C-terminal end, either:

-   a) one of the amino acid sequences given as SEQ ID NO's: 23 to 40 in    Table 3;

TABLE 3 FW4 sequences with C-terminal extension WGQGTQVTVKS(X)_(n)(SEQ ID NO: 23) WGKGTLVTVKS(X)_(n) (SEQ ID NO: 24) RGQGTRVTVKS(X)_(n)(SEQ ID NO: 25) WGLGTQVTISS(X)_(n) (SEQ ID NO: 26) GSQGTQVTVKS(X)_(n)(SEQ ID NO: 27) LRGGTQVTVKS(X)_(n) (SEQ ID NO: 28) RGQGTLVTVKS(X)_(n)(SEQ ID NO: 29) RSRGIQVTVKS(X)_(n) (SEQ ID NO: 30) WGKGTQVTVKS(X)_(n)(SEQ ID NO: 31) WGQGTQVTVQS(X)_(n) (SEQ ID NO: 32) WGKGTLVTVQS(X)_(n)(SEQ ID NO: 33) RGQGTRVTVQS(X)_(n) (SEQ ID NO: 34) WGLGTQVTISS(X)_(n)(SEQ ID NO: 35) GSQGTQVTVQS(X)_(n) (SEQ ID NO: 36) LRGGTQVTVQS(X)_(n)(SEQ ID NO: 37) RGQGTLVTVQS(X)_(n) (SEQ ID NO: 38) RSRGIQVTVQS(X)_(n)(SEQ ID NO: 39) WGKGTQVTVQS(X)_(n) (SEQ ID NO: 40)

-   -   in which (i) the amino acid residues of the FW4 sequences that        precede the (X)_(n) C-terminal extension in SEQ ID NO's: 23 to        40 correspond to the amino acid positions of FW4 of a VH domain        (i.e. positions 103 to 113 according to the Kabat        numbering); (ii) n is 1 to 10, preferably 1 to 5, such as 1, 2,        3, 4 or 5 (and preferably 1 or 2, such as 1); and (iii) each X        is an (preferably naturally occurring) amino acid residue that        is independently chosen, and preferably independently chosen        from the group consisting of alanine (A), glycine (G), valine        (V), leucine (L) or isoleucine (I); or

-   b) an amino acid sequence that has no more than three, preferably no    more than two, amino acid differences with at least one of the amino    acid sequences SEQ ID NO's 23 to 40 (in which said amino acid    differences are at the positions that correspond to the amino acid    positions of FW4 of a VH domain, i.e. at positions 103 to 113    according to the Kabat numbering, with any amino acid differences    within the C-terminal extension (X)_(n) being disregarded), in    which: (i) the amino acid residue at the position corresponding to    position 112 of the Kabat numbering is either K or Q; (ii) n is 1 to    10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or    2, such as 1); and (iii) each X is an (preferably naturally    occurring) amino acid residue that is independently chosen, and    preferably independently chosen from the group consisting of alanine    (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Again,    further to features (i) to (iii) as mentioned in the previous    sentence, in such an amino acid sequence: (iv) the amino acid    residue at the position corresponding to position 103 of the Kabat    numbering is preferably W or R; (v) the amino acid residue at the    position corresponding to position 104 of the Kabat numbering is    preferably G; (vi) the amino acid residue at the position    corresponding to position 106 of the Kabat numbering is preferably    G; (vii) the amino acid residue at the position corresponding to    position 107 of the Kabat numbering is preferably T; (viii) the    amino acid residue at the position corresponding to position 108 of    the Kabat numbering is preferably Q or L (and in humanized    nanobodies preferably L); (ix) the amino acid residue at the    position corresponding to position 109 of the Kabat numbering is    preferably V; (x) the amino acid residue at the position    corresponding to position 110 of the Kabat numbering is preferably T    (or alternatively may be K or Q); (xi) the amino acid residue at the    position corresponding to position 111 of the Kabat numbering is    preferably V; and reference is again made to the Table 2 for    possible amino acid residues that can be present at each position.

Preferably, according to this aspect of the invention, a VH domain ofthe invention has, at its C-terminal end, either:

-   a) an amino acid sequence that is WGQGTQVTVKS(X)_(n) (SEQ ID NO:23)    or WGQGTQVTVQS(X)_(n) (SEQ ID NO:32), in which (ii) n is 1 to 10,    preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2,    such as 1); and (iii) each X is an (preferably naturally occurring)    amino acid residue that is independently chosen, and preferably    independently chosen from the group consisting of alanine (A),    glycine (G), valine (V), leucine (L) or isoleucine (I); or-   b) an amino acid sequence that has no more than three, preferably no    more than two, amino acid differences with at least one of the amino    acid sequences WGQGTQVTVKS(X)_(n) (SEQ ID NO:23) or    WGQGTQVTVQS(X)_(n) (SEQ ID NO:32) (in which said amino acid    differences are at the positions that correspond to the amino acid    positions of FW4 of a VH domain, i.e. at positions 103 to 113    according to the Kabat numbering, with any amino acid differences    within the C-terminal extension (X)_(n) being disregarded), in    which: (i) the amino acid residue at the position corresponding to    position 112 of the Kabat numbering is either K or Q; (ii) n is 1 to    10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or    2, such as 1); and (iii) each X is an (preferably naturally    occurring) amino acid residue that is independently chosen, and    preferably independently chosen from the group consisting of alanine    (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Again,    for such amino acid sequences, features (vi) to (xi) as described in    the preceding paragraphs preferably also apply.

Also, as mentioned herein, the VH domains of the invention can alsocontain other amino acid residues or substitutions that are known per sein the art for VH domains and in particular for ISVD's (and more inparticular for Nanobodies) at the relevant positions. Some non-limitingexamples may be as mentioned herein and for example include one or moreof the “Hallmark residues” that are characteristic of VHH's/Nanobodies(including for example a leucine (L) at position 11), other amino acidresidues that naturally occur in VHH's (such as an alanine (A) atposition 14), humanizing substitutions known per se for VHH's/Nanobodies(such as Q108L and A14P), one or more of the mutations suggested byHarmsen (such as V89M or V89T) and/or at the positions suggested byNieba (e.g. 11, 87 or 89); or any suitable combination thereof; and/orfor example a T110K, T110Q or V89L mutation.

When a VH domain of the invention contains a C-terminal extension(X)_(n), according to some preferred, but non-limiting examples of suchextensions, X and n can be as follows:

-   (a) n=1 and X=Ala;-   (b) n=2 and each X=Ala;-   (c) n=3 and each X=Ala;-   (d) n=2 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (e) n=3 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (f) n=3 and at least two X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (g) n=1 and X=Gly;-   (h) n=2 and each X=Gly;-   (i) n=3 and each X=Gly;-   (j) n=2 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (k) n=3 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (l) n=3 and at least two X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (m) n=2 and each X=Ala or Gly;-   (n) n=3 and each X=Ala or Gly;-   (o) n=3 and at least one X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile); or-   (p) n=3 and at least two X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);    with aspects (a), (b), (c), (g), (h), (i), (m) and (n) being    particularly preferred, with aspects in which n=1 or 2 being    preferred and aspects in which n=1 being particularly preferred.

It should also be noted that, preferably, any C-terminal extensionpresent in a VH domain of the invention does not contain a (free)cysteine residue (unless said cysteine residue is used or intended forfurther functionalization, for example for pegylation).

Also, the preferences that are indicated on pages 35 to 41 of WO12/175741 for the C-terminal extensions used according to WO 12/175741(which are also the preferred C-terminal extensions used in the VHdomains according to the present invention) also apply to the C-terminalextensions used in the VH domains of the inventions, and thesepreferences according to WO 12/175741 are also included herein byreference.

Preferably, when a VH domain of the invention contains a C-terminalextension (X)_(n), n=1, 2 or 3, and each X is either Ala of Gly. Morepreferably, each X is Ala, and n=1 or 2, and preferably 1.

When the VH domains of the invention contain a C-terminal extension,they will usually be present at (and often form) the C-terminal end ofthe protein, polypeptide, compound, construct or other chemical entityin which it is present. Again, such a protein, polypeptide, compound,construct or other chemical entity may contain one or more other VHdomains of the invention (i.e. not at the C-terminal end); in such acase, said other VH domains of the invention will contain a lysine (K)or glutamine (Q) at position 112 (and be as further described herein),but will not contain a C-terminal extension (instead, they may be linked(optionally via one or more suitable linkers) at their C-terminus to oneor more other amino acid sequences, moieties, binding domains or bindingunits present in the protein, polypeptide, compound, construct or otherchemical entity, such as the VH domain of the invention with theC-terminal extension that is present at the C-terminal end).

As the VH domains of the invention (and the proteins, polypeptides,compounds, constructs and other chemical entities comprising the same asfurther described herein) are particularly useful in (and intended for)use pharmaceutical uses (such as the prevention, treatment and/ordiagnosis of diseases and disorders in human subjects in need of thesame), they preferably have a high degree of sequence homology in theirframework regions with the framework sequences of human VH domains. Inparticular, the VH domains of the invention preferably have an overalldegree of sequence identity (determined as further described herein, andtaking into account only the framework regions and not the CDR's, andalso not taking into account the substitution at position 112 and anyC-terminal extension if present) with at least one human germlinesequence (such as DP-47, DP-51 or DP-29) of at least 80%, preferably atleast 85%, such as 90% or more. More in particular, the VH domains ofthe invention preferably have an overall degree of sequence identity(determined as further described herein, and taking into account onlythe framework regions and not the CDR's, and also not taking intoaccount the substitution at position 112 and any C-terminal extension ifpresent) of at least 80%, preferably at least 85%, such as 90% or morewith at least one of the following human germline sequences: DP-47,DP-51 and/or DP-29.

As further described herein, according to one aspect of the invention,the VH domain of the invention can be a heavy-chain variable domainthat, in the protein, polypeptide, protein or construct in which is itpresent, interacts/associates (or is intended to interact/associate)with a VL domain in order to form an antigen binding site, in which atleast the VH domain has an exposed C-terminal end or region. Forexample, a VH domain according to this aspect of the invention can be aVH domain that is present and/or used in an ScFv and or a diabody, whereit will associate with a VL domain to form an antigen binding site.

However, according to a preferred aspect of the invention, the VH domainof the invention is an (heavy-chain) immunoglobulin single variabledomain or “ISVD”, meaning a heavy-chain variable domain that can form afunctional antigen binding site without interaction with a VL domain.For example, the VH domain of the invention can be a Nanobody (includinga VHH, a humanized VHH and/or a camelized VHs such as camelized humanVH's), a (single domain) antibody is a VH domain or that is derived froma VH domain, or a dAb that is a VH domain or that is derived from a VHdomain. The VH domain of the invention is preferably a nanobody (andmore preferably VHH domain, a humanized VHH domain or a camelized VHdomain such as a camelized human VH domain).

In the present specification:

-   -   the term “Nanobody” is generally as defined in or WO 08/020079        or WO 09/138519, and thus in a specific aspect generally denotes        a VHH, a humanized VHH or a camelized VH (such as a camelized        human VH) or generally a sequence optimized VHH (such as e.g.        optimized for chemical stability and/or solubility, maximum        overlap with known human framework regions and maximum        expression). It is noted that the terms Nanobody or Nanobodies        are registered trademarks of Ablynx N.V. and thus may also be        referred to as Nanobody® and/or Nanobodies®);    -   the term “ISVD” (or “ISV”) as used herein in its broadest sense        also includes “ISVD-based biologicals” and, when the ISVD is a        Nanobody, “Nanobody-based biologicals”. An “ISVD-based        biological” is defined herein as a protein, polypeptide or other        biological drug that comprises or essentially consist of at        least one (such as one, two or three) ISVD's. Similarly, a        “Nanobody-based biological” is defined as a protein, polypeptide        or other biological drug that comprises or essentially consist        of at least one (such as one, two or three) Nanobodies. As with        the term “ISVD”, whenever the term “ISVD-based biological” is        used, it should be understood that such an ISVD-based biological        is preferably a Nanobody-based biological. Within the context of        the present invention, both an “ISVD-based biological” and a        “Nanobody-based biological” may for example be a monovalent,        bivalent (or multivalent), bispecific (or multispecific), and        biparatopic (or “multiparatopic) ISVD construct or Nanobody        construct, respectively. Also, any ISVD-based or Nanobody-based        biological may for example, in addition to the one or more (such        as one, two or three) ISVD's or Nanobodies, optionally further        comprise one or more (such as one or two) other further        therapeutic moieties and/or one or more (such as one or two)        other moieties that influence the pharmacokinetic or        pharmacodynamic properties of the ISVD-based or Nanobody-based        biological (such as its half-life). Suitable examples of such        further therapeutic or other moieties will be clear to the        skilled person, and for example generally can include any        therapeutically active protein, polypeptide or other binding        domain or binding unit, as well as for example modifications        such as those described on pages 149 to 152 of WO 09/138159. An        ISVD-based biological or Nanobody-based biological is preferably        a therapeutic or intended for use as a therapeutic (which        includes prophylaxis and diagnosis) and for this purpose        preferably contains at least one ISVD against a therapeutically        relevant target (such as for example RANK-L, vWF, IgE, RSV,        CXCR4, IL-23 or other interleukins, etc.). For some specific but        non-limiting examples of such ISVD-based or Nanobody-based        biologicals, reference is to Examples 8 to 18 and also for        example made to the various applications by Ablynx N.V. (such as        for example and without limitation WO 2004/062551, WO        2006/122825, WO 2008/020079 and WO 2009/068627), as well as for        example (and without limitation) to applications such as WO        06/038027, WO 06/059108, WO 07/063308, WO 07/063311, WO        07/066016 and WO 07/085814. Also, as further described herein,        an ISVD or Nanobody as described herein may be directed against        a (human) serum protein such as (human) serum albumin, and such        an ISVD or Nanobody may also find therapeutic uses, in        particular in and/or for extending the half-life of therapeutic        moieties and compounds (such as in or for the IS V-based        biologicals described herein). Reference is for example made to        WO 2004/041865, WO 2006/122787 and WO 2012/175400, which        generally describe the use of serum-albumin binding nanobodies        for half-life extension. Also, in the present specification,        unless explicitly mentioned otherwise herein, all terms        mentioned herein have the meaning given in WO 09/138519 (or in        the prior art cited in WO 09/138519) or WO 08/020079 (or in the        prior art cited in WO 08/020079). Also, where a method or        technique is not specifically described herein, it can be        performed as described in WO 09/138519 (or in the prior art        cited in WO 09/138519) or WO 08/020079 (or in the prior art        cited in WO 08/020079).

Also, when used in the present specification or claims, the followingterms have the same meaning as given on, and/or where applicable can bedetermined in the manner described in, pages 62-75 of WO 09/138519:“agonist”, “antagonist”, “inverse agonist”, “non-polar, uncharged aminoacid residue”, “polar uncharged amino acid residue”, “polar, chargedamino acid residue”, “sequence identity”, “exactly the same” and “aminoacid difference” (when referring to a sequence comparison of two aminoacid sequences), “(in) essentially isolated (form)”, “domain”, “bindingdomain”, “antigenic determinant”, “epitope”, “against” or “directedagainst” (an antigen), “specificity” and “half-life”. In addition, theterms “modulating” and “to modulate”, “interaction site”, “specificfor”, “cross-block”, “cross-blocked” and “cross-blocking” and“essentially independent of the pH” are as defined on (and/or can bedetermined as described on) pages 74-79 of WO 10/130832 of applicant.Also, when referring to a construct, compound, protein or polypeptide ofthe invention, terms like “monovalent”, “bivalent” (or “multivalent”),“bispecific” (or “multispecific”), and “biparatopic” (or“multiparatopic”) may have the meaning given in WO 09/138.519, WO10/130832 or WO 08/020079.

The term “half-life” as used herein relation to an ISVD, Nanobody,ISVD-based biological, Nanobody-based biological or any other amino acidsequence, compound or polypeptide referred to herein can generally bedefined as described in paragraph o) on page 57 of WO 08/020079 and asmentioned therein refers to the time taken for the serum concentrationof the amino acid sequence, compound or polypeptide to be reduced by50%, in vivo, for example due to degradation of the sequence or compoundand/or clearance or sequestration of the sequence or compound by naturalmechanisms. The in vivo half-life of an amino acid sequence, compound orpolypeptide of the invention can be determined in any manner known perse, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may for example generally beas described in paragraph o) on page 57 of WO 08/020079. As alsomentioned in paragraph o) on page 57 of WO 08/020079, the half-life canbe expressed using parameters such as the t½-alpha, t½-beta and the areaunder the curve (AUC). In this respect it should be noted that the term“half-life” as used herein in particular refers to the t½-beta orterminal half-life (in which the t½-alpha and/or the AUC or both may bekept out of considerations). Reference is for example made to theExperimental Part below, as well as to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).Similarly, the terms “increase in half-life” or “increased half-life” asalso as defined in paragraph o) on page 57 of WO 08/020079 and inparticular refer to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

When a term is not specifically defined herein, it has its usual meaningin the art, which will be clear to the skilled person. Reference is forexample made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10th Ed. Blackwell Publishing, U K (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, N.Y. (2005), as well as to the generalbackground art cited herein.

Also, as already indicated herein, the amino acid residues of a Nanobodyare numbered according to the general numbering for VH domains given byKabat et al. (“Sequence of proteins of immunological interest”, USPublic Health Services, NIH Bethesda, Md., Publication No. 91), asapplied to VHH domains from Camelids in the article of Riechmann andMuyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195; orreferred to herein. According to this numbering, FR1 of a Nanobodycomprises the amino acid residues at positions 1-30, CDR1 of a Nanobodycomprises the amino acid residues at positions 31-35, FR2 of a Nanobodycomprises the amino acids at positions 36-49, CDR2 of a Nanobodycomprises the amino acid residues at positions 50-65, FR3 of a Nanobodycomprises the amino acid residues at positions 66-94, CDR3 of a Nanobodycomprises the amino acid residues at positions 95-102, and FR4 of aNanobody comprises the amino acid residues at positions 103-113. [Inthis respect, it should be noted that—as is well known in the art for VHdomains and for VHH domains—the total number of amino acid residues ineach of the CDR's may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence. Generally, however, it can be said that, according tothe numbering of Kabat and irrespective of the number of amino acidresidues in the CDR's, position 1 according to the Kabat numberingcorresponds to the start of FR1 and vice versa, position 36 according tothe Kabat numbering corresponds to the start of FR2 and vice versa,position 66 according to the Kabat numbering corresponds to the start ofFR3 and vice versa, and position 103 according to the Kabat numberingcorresponds to the start of FR4 and vice versa.].

Alternative methods for numbering the amino acid residues of VH domains,which methods can also be applied in an analogous manner to VHH domainsfrom Camelids and to Nanobodies, are the method described by Chothia etal. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and theso-called “contact definition”. However, in the present description,aspects and figures, the numbering according to Kabat as applied to VHHdomains by Riechmann and Muyldermans will be followed, unless indicatedotherwise.

It should also be noted that the Figures, any Sequence Listing and theExperimental Part/Examples are only given to further illustrate theinvention and should not be interpreted or construed as limiting thescope of the invention and/or of the appended claims in any way, unlessexplicitly indicated otherwise herein.

The VH domains of the invention can be directed against any suitable ordesired antigen or target, including any pharmaceutically and/ortherapeutically relevant target as described herein.

According to one specific aspect of the invention, the VH domains of theinvention are directed against a serum protein, and in particular ahuman serum protein. According to a preferred aspect, when a VH domainof the invention is directed against a serum protein, it is directedagainst serum albumin, and in particular human serum albumin. Thus, theinvention also relates to a VH domain of the invention (as definedherein, including the preferences defined for the VH domains of theinvention) that can specifically bind to a serum protein, in particulara human serum protein, and that can preferably specifically bind toserum albumin, and more preferably to human serum albumin. Again, such aVH domain is preferably an ISVD (as described herein) and morepreferably a Nanobody.

For example, a VH domain of the invention against serum albumin can beone of the Nanobodies against (human) serum albumin that are describedin WO 2004/041865, and in particular in WO 2006/122787 and WO2012/175400 (all applications from applicant/assignee), in which theamino acid at position 112 is substituted by either K or Q, and which isoptionally provided with a C-terminal extension as described herein (andcan also for example also contain one or more of the other specificamino acid residues/substitutions mentioned herein, such as 11L, L11V,L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q,T110Q, 110K and/or T110K; although usually the presence of an additionalsubstitution at position 110 will not often not be required whenposition 112 is K or Q, in which case position 110 is preferably T).Furthermore, it is envisaged that the present invention can also beapplied to other serum-albumin binding heavy-chain ISVD's, such as thosedescribed in WO 03/035694, WO 04/003019, WO 05/118642, WO 06/059106, WO08/096158, WO 09/121804WO 10/108937 or US 2013/0129727, i.e. by suitablyintroducing the substitutions described herein (i.e. at least one ofS112K, S112Q and/or V89T, and optionally one or more of the other aminoacid residues/substitutions described herein, such as L11V) andoptionally (and usually preferably as outlined herein) adding aC-terminal extension (as further described herein). Some preferred butnon-limiting examples of such serum albumin binding Nanobodies of theinvention are humanized variants of the amino acid sequence of SEQ IDNO: 52 of WO 2006/122787 (called “Alb-1” in WO 2006/122787), in whichthe amino acid at position 112 is substituted by either K or Q (andwhich is optionally provided with a C-terminal extension as describedherein), such as the humanized variants of Alb-1 that are given in SEQID NO's: 57 to 64 of WO 2006/122787 (in each case, with a S112K or S112Qsubstitution, and optionally with a C-terminal extension) or thehumanized variants of Alb-1 that are given in SEQ ID NO's 3 to 11 of WO2012/175400 (again, in each case, with a S112K or S112Q substitution),of which SEQ ID NO's 3, 4 and 5 can optionally contain a C-terminalextension, and SEQ ID NO's 6 to 11 already contain a C-terminalextension (and again, such variants can contain one or more of the otherspecific amino acid residues/substitutions mentioned herein, such as11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L,Q108L, 110Q, T110Q, 110K and/or T110K; although usually the presence ofan additional substitution at position 110 will not often not berequired when position 112 is K or Q, in which case position 110 ispreferably T). Also: (i) position 41 may for example be one of the aminoacid residues (i.e. human VH3 residues and/or Camelid VHH residues)mentioned in Table A-6 of WO 08/020079 for position 41 and may inparticular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

Thus, in a further aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

-   -   the amino acid residue at position 112 is either K or Q;        and that optionally contains at its C-terminal end a C-terminal        extension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such        as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each        X is an (preferably naturally occurring) amino acid residue that        is independently chosen, and preferably independently chosen        from the group consisting of alanine (A), glycine (G), valine        (V), leucine (L) or isoleucine (I) (again, such a C-terminal        extension is preferably as further described herein, and again        such a Nanobody can also for example also contain one or more of        the other specific amino acid residues/substitutions mentioned        herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T,        V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and/or T110K;        although usually the presence of an additional substitution at        position 110 will not often not be required when position 112 is        K or Q, in which case position 110 is preferably T). Also: (i)        position 41 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-6 of WO 08/020079 for position 41 and may in        particular be (or be chosen from) proline (P), serine (S),        threonine (T), alanine (A) or leucine (L), which are some of the        amino acid residues that most frequently occur at this position        in either humans or llamas, and may more in particular be either        proline (P) or alanine (A); and/or (ii) position 42 may for        example be one of the amino acid residues (i.e. human VH3        residues and/or Camelid VHH residues) mentioned in Table A-6 of        WO 08/020079 for position 42 and may in particular be (or be        chosen from) glycine (G) or glutamic acid (E); and/or (iii)        position 87 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-7 of WO 08/020079 for position 87 and may in        particular be (or be chosen from) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that is a humanized variant of SEQ ID NO: 52 of WO 2006/122787in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

-   -   the amino acid residue at position 112 is either K or Q;        and that optionally contains at its C-terminal end a C-terminal        extension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such        as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each        X is an (preferably naturally occurring) amino acid residue that        is independently chosen, and preferably independently chosen        from the group consisting of alanine (A), glycine (G), valine        (V), leucine (L) or isoleucine (I) (again, such a C-terminal        extension is preferably as further described herein, and again        such a Nanobody can also for example also contain one or more of        the other specific amino acid residues/substitutions mentioned        herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T,        V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and/or T110K;        although usually the presence of an additional substitution at        position 110 will not often not be required when position 112 is        K or Q, in which case position 110 is preferably T). Also: (i)        position 41 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-6 of WO 08/020079 for position 41 and may in        particular be (or be chosen from) proline (P), serine (S),        threonine (T), alanine (A) or leucine (L), which are some of the        amino acid residues that most frequently occur at this position        in either humans or llamas, and may more in particular be either        proline (P) or alanine (A); and/or (ii) position 42 may for        example be one of the amino acid residues (i.e. human VH3        residues and/or Camelid VHH residues) mentioned in Table A-6 of        WO 08/020079 for position 42 and may in particular be (or be        chosen from) glycine (G) or glutamic acid (E); and/or (iii)        position 87 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-7 of WO 08/020079 for position 87 and may in        particular be (or be chosen from) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that has at least 80%, preferably at least 85%, more preferablyat least 90% such as at least 95% sequence identity with at least one ofAlb-1 (SEQ ID NO: 52 of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein)and/or Alb-23 (SEQ ID NO:61 herein) (taking into account both theframework sequences and the CDR's, but not any C-terminal extension), inwhich the amino acid residue at position 112 is either K or Q, and thatoptionally contains at its C-terminal end a C-terminal extension (X)_(n)in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, sucha C-terminal extension is preferably as further described herein, andagain such a Nanobody can also for example also contain one or more ofthe other specific amino acid residues/substitutions mentioned herein,such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L,108L, Q108L, 110Q, T110Q, 110K and/or T110K; although usually thepresence of an additional substitution at position 110 will not oftennot be required when position 112 is K or Q, in which case position 110is preferably T). Also: (i) position 41 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

Such a Nanobody of the invention is again preferably a humanized variantof Alb-1 (but with a S112K or S112Q substitution), and more preferablyhas at least one, in particular any two, and more in particular allthree of CDR1, CDR2 and/or CDR3 given in SEQ ID NO's 41 to 43,respectively.

According to one specific aspect, any serum albumin-binding Nanobody ofthe invention can also have the amino acid residues that arecharacteristic of Alb-23 and its variants as described in WO 12/175400(i.e. the amino acid motif GP on positions 44 and 45, the amino acidmotif SKN on positions 74 to 76, and preferably a G at position 16 andoptionally also an R at position 83).

Some preferred, but non-limiting examples of Nanobodies of the inventionthat are directed against human serum albumin are given in Table 4 andExample 20.

TABLE 4Non-limiting examples of VH domains of the invention directed against human serum albumin.Amino Acid at SEQ Sequence position C-terminal ID: name 112 extensionSequence 46 Alb-11 S [none]EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS (WO (reference)GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR 06/122787)SSQGTLVTVSS 47 Alb-11 + K [none]EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVKS 48Alb-11 + K A EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112K +GSGSDTLYADSVKGRFT1SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR A SSQGTLVTVKSA 49Alb-11 + K AA EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS112K + GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR AASSQGTLVTVKSAA 50 Alb-11 + K AAAEVOLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112K +GSGSDTLYADSVKGRFT1SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR AAA SSQGTLVTVKSAAA51 Alb-11 + K G EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS112K + GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR GSSQGTLVTVKSG 52 Alb-11 + K GGEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112K +GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR GG SSQGTLVTVKSGG 53Alb-11 K GGG EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112K +GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR GGG SSQGTLVTVKSGGG54 Alb-11 + Q [none]EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVQS 55Alb-11 + Q A EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112Q +GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR A SSQGTLVTVQSA 56Alb-11 + Q AA EVQLVESGGGLVQPGNSLRLSCAASGPTFSSFGMSWVRQAPGKGLEWVSSIS112Q + GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR AASSQGTLVTVQSAA 57 Alb-11 + Q AAAEV0LVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVR0APGKGLEWVSSIS 112Q +GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR AAA SSQGTLVTVQSAAA58 Alb-11 + Q G EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS112Q + GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEOTAVYYCTIGGSLSR GSSQGTLVTVQSG 59 Alb-11 + Q GGEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 112Q +GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR GG SSQGTLVTVQSGG 60Alb-11 + Q GGG EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS112Q + GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR GGGSSQGTLVTVQSGGG 61 Alb-23 S [none]EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS (WO 12/ (reference)GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR 175400) SSQGTLVTVSS62 Alb-23 + K [none]EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVKS 63Alb-23 + K A EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +A SSQGTLVTVKSA 64Alb-23 + K AA EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +AA SSQGTLVTVKSAA65 Alb-23 + K AAA EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS112K GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +AAASSQGTLVTVKSAAA 66 Alb-23 + K GEVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +G SSQGTLVTVKSG 67Alb-23 + K GG EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112KGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +GG SSQGTLVTVKSGG68 Alb-23 + K GGG EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS112K GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +GGGSSQGTLVTVKSGGG 69 Alb-23 + Q [none]EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVQS 70Alb-23 + Q A EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +A SSQGTLVTVQSA 71Alb-23 + Q AA EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +AA SSQGTLVTVQSAA72 Alb-23 + Q AAA EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS112Q GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +AAASSQGTLVTVQSAAA 73 Alb-23 + Q GEVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +G SSQGTLVTVQSG 74Alb-23 + Q GG EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS 112QGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +GG SSQGTLVTVQSGG75 Alb-23 + Q GGG EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSIS112Q GSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSR +GGGSSQGTLVTVQSGGG

In another aspect, the invention relates to a VH domain, and inparticular an ISVD, and more in particular an Nanobody, that has:

-   a) a FW 4 sequence that is one of the following FW4 sequences:

TABLE 5 FW4 sequences SSQGTLVTVKS (SEQ ID NO: 99) SSQGTLVTVQS(SEQ ID NO: 100) SSQGTLVKVSS (SEQ ID NO: 101) SSQGTLVQVSS(SEQ ID NO: 102) SSQGTLVTVKS(X)_(n) (SEQ ID NO: 103) SSQGTLVTVQS(X)_(n)(SEQ ID NO: 104) SSQGTLVKVSS(X)_(n) (SEQ ID NO: 105) SSQGTLVQVSS(X)_(n)(SEQ ID NO: 106)

-   -   in which (i) the amino acid residues of the FW4 sequences that        precede the (X)_(n) C-terminal extension in SEQ ID NO's: 23 to        40 correspond to the amino acid positions of FW4 of a VH domain        (i.e. positions 103 to 113 according to the Kabat        numbering); (ii) n is 1 to 10, preferably 1 to 5, such as 1, 2,        3, 4 or 5 (and preferably 1 or 2, such as 1); and (iii) each X        is an (preferably naturally occurring) amino acid residue that        is independently chosen, and preferably independently chosen        from the group consisting of alanine (A), glycine (G), valine        (V), leucine (L) or isoleucine (I); or

-   b) an amino acid sequence that has no more than three, preferably no    more than two, amino acid differences with at least one of the amino    acid sequences SEQ ID NO's: 99 to 106 (in which said amino acid    differences are at the positions that correspond to the amino acid    positions of FW4 of a VH domain, i.e. at positions 103 to 113    according to the Kabat numbering, with any amino acid differences    within the C-terminal extension (X)_(n) being disregarded), in    which: (i) when the amino acid residue at the position corresponding    to position 112 of the Kabat numbering is either K or Q, said amino    acid differences are at another amino acid position than 112, and    the amino acid residue at position 89 of the VH domain is preferably    chosen from V, T or L (and is most preferably V); (i) when the amino    acid residue at the position corresponding to position 110 of the    Kabat numbering is either K or Q, then said amino acid differences    are at another amino acid position than 110, and position 89 is the    VH domain is L; (iii) n is 1 to 10, preferably 1 to 5, such as 1, 2,    3, 4 or 5 (and preferably 1 or 2, such as 1); and (iv) each X is an    (preferably naturally occurring) amino acid residue that is    independently chosen, and preferably independently chosen from the    group consisting of alanine (A), glycine (G), valine (V),    leucine (L) or isoleucine (I). Again, further to features (i)    to (iv) as mentioned in the previous sentence, in such an amino acid    sequence: (v) the amino acid residue at the position corresponding    to position 103 of the Kabat numbering is preferably S; (vi) the    amino acid residue at the position corresponding to position 104 of    the Kabat numbering is preferably S; (vii) the amino acid residue at    the position corresponding to position 106 of the Kabat numbering is    preferably G; (viii) the amino acid residue at the position    corresponding to position 107 of the Kabat numbering is preferably    T; (ix) the amino acid residue at the position corresponding to    position 108 of the Kabat numbering is preferably Q or L (and in    humanized nanobodies preferably L); (ix) the amino acid residue at    the position corresponding to position 109 of the Kabat numbering is    preferably V; and reference is again made to the Table 2 for    possible amino acid residues that can be present at each position.

Again, in such VH domains of the invention: (a) the amino acid residueat position 11 is one of L, V or K (and more preferably V); the aminoacid residue at position 14 is preferably one of A or P; the amino acidresidue at position 41 is preferably one of A or P. Also, such a VHdomain of the invention is preferably an ISVD, and more preferably aNanobody; and may again be directed against human serum albumin (inwhich case it preferably has CDR1, CDR2 and CDR3 respectively thatcorrespond to SEQ ID NO's 41, 42 and 43, respectively). Also, when saidVH domains are ISVD's or nanobodies against human serum albumin, theycan further be as described herein for nanobodies of the invention thatare directed against human serum albumin.

The VH domains of the invention that are directed against serum albumin(and in particular a serum-albumin binding ISVD of the invention) can beused to increase the half-life of therapeutically active compounds,(poly)peptides, proteins, binding domains, binding units or othertherapeutically active entities or moieties, essentially in the mannerdescribed in WO 2004/041865, WO 2006/122787 and/or WO 2012/175400 forthe use of the serum albumin-binding nanobodies that are disclosed insaid references (i.e. by suitably linking the serum albumin-binding ISVDto the protein, polypeptide, compound or other entities, optionally viaa suitable linker. For example, WO 12/175400 on pages 12 and 13 givessome examples of the manner in which suitable fusion proteins can beconstructed).

In another aspect, the invention relates to an immunoglobulinheavy-chain variable domain (VH domain) in which the amino acid residueat position 89 (Kabat numbering) is threonine (T) and the amino acidresidue at position 112 is either a serine (S), lysine (K) residue orglutamine (Q) residue. Such immunoglobulin heavy-chain variable domains(VH domain) with a T at position 89 are also included in the term “VHdomains of the invention” as used herein in its broadest sense and canfurther be as described herein for the VH domains of the invention thatcomprise a K or Q at position 112. Accordingly, such an immunoglobulinheavy-chain variable domain (VH domain) can have a C-terminal extensionas further described herein (including the indicated preferences forsuch a C-terminal extension); can be an ISVD and in particular aNanobody as further described herein.

Again, if such an VH domain of the invention has an exposed C-terminalregion (for example, because it forms the C-terminal end of the protein,polypeptide or other construct in which it is present), it preferablycontains a C-terminal extension (reference is made to the data shown inTable C below).

Also, Nanobodies of the invention in which position 89 is T (i.e. withor without a C-terminal extension) preferably contain an amino acid atposition 11 that is chosen from L (the most often occurring amino acidresidue in VHH's), E, K, M, S, V, W or Y; more preferably from L, E, K,V or Y, and even more preferably from L, K or V (with V being mostpreferred). For example, they can contain an L11K or L11V substitution,as well as for example a P14A or A14P substitution, a Q108Lsubstitution, and/or a T110K, T110Q, S112K and/or S112Q substitution(although usually the presence of one or two additional substitutions atposition 110 and/or 112 will not often not be required when position 89is T, in which case position 110 is preferably T and position 112 ispreferably S).

In particular, an immunoglobulin heavy-chain variable domain (VH domain)of the invention according to this particular aspect has a T at position89 (Kabat) and a C-terminal end that is one of VTVSS (SEQ ID NO:77),VTVSS(X)n (SEQ ID NO:78), VTVKS (SEQ ID NO:1), VTVKS(X)n (SEQ ID NO:21),VTVQS (SEQ ID NO:2), VTVQS(X)n (SEQ ID NO:22), VKVSS (SEQ ID NO: 95),VKVSS(X)n (SEQ ID NO:97), VQVSS (SEQ ID NO: 96), VQVSS(X)n (SEQ ID NO:98), VZVZS (SEQ ID NO: 107, in which each amino acid residue Z isindependently K or Q) or VZVZSX(n) (SEQ ID NO: 108, in which each aminoacid residue Z is independently K or Q) (and in particular one of VTVKS(SEQ ID NO:1), VTVQS (SEQ ID NO:2), VTVSS (SEQ ID NO:77), VTVKS(X)_(n)(SEQ ID NO:21), VTVQS(X)_(n) (SEQ ID NO:22) or VTVSS(X)_(n) (SEQ IDNO:78), and more in particular be either VTVSS (SEQ ID NO:77) orVTVSS(X)n (SEQ ID NO:78)), in which n and X are as further describedherein for the VH domains of the invention in which position 112 is Q orK (and in which any C-terminal extension is preferably as furtherdescribed herein for the VH domains of the invention in which position112 is Q or K). Also, as is the case for the VH domains of the inventionin which position 112 is Q or K, when such a VH domain with a T atposition 89 is a nanobody, position 11 is preferably a leucine (L),position 14 can in particular be alanine (A) or proline (P) and position108 can in particular be Q or L (and in humanized nanobodies preferablyL); and such a Nanobody with a T at position 89 can contain one or morenanobody hallmark residues and/or can be suitably humanized. Also: (i)position 41 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 41 and may in particular be (or be chosenfrom) proline (P), serine (S), threonine (T), alanine (A) or leucine(L), which are some of the amino acid residues that most frequentlyoccur at this position in either humans or llamas, and may more inparticular be either proline (P) or alanine (A); and/or (ii) position 42may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 42 and may in particular be (or be chosen from)glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

Such a VH domain of the invention with a T at position 89 can again alsobe a serum albumin Nanobody as further described herein. For example,such a serum albumin-binding Nanobody can be one of the sequences of SEQID NOs: 46 to 75, but with a T at position 89; or another serum albuminbinding Nanobody with a T at position 89 that has at least 80%,preferably at least 85%, more preferably at least 90% such as at least95% sequence identity with at least one of Alb-1 (SEQ ID NO: 52 of WO2006/122787), Alb-8 (SEQ ID NO: 46 herein) and/or Alb-23 (SEQ ID NO:61herein) (taking into account both the framework sequences and the CDR's,but not any C-terminal extension). Some other examples of suchNanobodies with a T at position 89 are given in SEQ ID NO's: 78 to 91(these are further variants of Alb-1/Alb-8 or Alb-23 with a T atposition 89 and an S at position 112).

More generally, a VH domain against serum albumin according to thisaspect of the invention can be one of the Nanobodies against (human)serum albumin that are described in WO 2004/041865, and in particular inWO 2006/122787 and WO 2012/175400 (all applications fromapplicant/assignee), in which the amino acid at position 89 is athreonine (T), and which is optionally provided with a C-terminalextension as described herein (and can also for example also suitablycontain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, S112K and/orS112Q; although usually the presence of additional substitutions atpositions 110 and/or 112 will not often not be required when a T ispresent at position 89, in which case that position 110 is preferably Tand position 112 is preferably S). Also: (i) position 41 may for examplebe one of the amino acid residues (i.e. human VH3 residues and/orCamelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A). Furthermore, it isenvisaged that the present invention can also be applied otherserum-albumin binding heavy-chain ISVD's, such as those described in WO03/035694, WO 04/003019, WO 05/118642, WO 06/059106, WO 08/096158, WO09/121804WO 10/108937 or US 2013/0129727, i.e. by suitably introducingthreonine (T) at position 89, and optionally one or more of the otheramino acid residues/substitutions described herein) and optionally (andusually preferably as outlined herein) adding a C-terminal extension (asfurther described herein).

Some preferred but non-limiting examples of such serum albumin bindingNanobodies of the invention are humanized variants of the amino acidsequence of SEQ ID NO: 52 of WO 2006/122787 (called “Alb-1” in WO2006/122787), in which the amino acid at position 89 is T (and which isoptionally provided with a C-terminal extension as described herein),such as the humanized variants of Alb-1 that are given in SEQ ID NO's:57 to 64 of WO 2006/122787 (in each case, with a V89T substitution, andoptionally with a C-terminal extension) or the humanized variants ofAlb-1 that are given in SEQ ID NO's 3 to 11 of WO 2012/175400 (again, ineach case, with a V89T substitution), of which SEQ ID NO's 3, 4 and 5can optionally contain a C-terminal extension, and SEQ ID NO's 6 to 11already contain a C-terminal extension (and again, such variants cancontain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K,S112Q and/or S112K; although usually the presence of one or twoadditional substitutions at positions 110 and/or 112 will not often notbe required when position 89 is T, in which case position 110 ispreferably T and position 112 is preferably S). Also: (i) position 41may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 41 and may in particular be (or be chosen from)proline (P), serine (S), threonine (T), alanine (A) or leucine (L),which are some of the amino acid residues that most frequently occur atthis position in either humans or llamas, and may more in particular beeither proline (P) or alanine (A); and/or (ii) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (iii) position 87 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).

Thus, in a further aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

-   -   the amino acid residue at position 89 is T;        and that optionally contains at its C-terminal end a C-terminal        extension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such        as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each        X is an (preferably naturally occurring) amino acid residue that        is independently chosen, and preferably independently chosen        from the group consisting of alanine (A), glycine (G), valine        (V), leucine (L) or isoleucine (I) (again, such a C-terminal        extension is preferably as further described herein, and again        such a Nanobody can also for example also contain one or more of        the other specific amino acid residues/substitutions mentioned        herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L,        Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q and/or S112K;        although usually the presence of one or two additional        substitutions at positions 110 and/or 112 will not often not be        required when position 89 is T, in which case position 110 is        preferably T and position 112 is preferably S). Also: (i)        position 41 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-6 of WO 08/020079 for position 41 and may in        particular be (or be chosen from) proline (P), serine (S),        threonine (T), alanine (A) or leucine (L), which are some of the        amino acid residues that most frequently occur at this position        in either humans or llamas, and may more in particular be either        proline (P) or alanine (A); and/or (ii) position 42 may for        example be one of the amino acid residues (i.e. human VH3        residues and/or Camelid VHH residues) mentioned in Table A-6 of        WO 08/020079 for position 42 and may in particular be (or be        chosen from) glycine (G) or glutamic acid (E); and/or (iii)        position 87 may for example be one of the amino acid residues        (i.e. human VH3 residues and/or Camelid VHH residues) mentioned        in Table A-7 of WO 08/020079 for position 87 and may in        particular be (or be chosen from) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that is a humanized variant of SEQ ID NO: 52 of WO 2006/122787in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

the amino acid residue at position 89 is T;

and that optionally contains at its C-terminal end a C-terminalextension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such as 1,2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (again, such a C-terminal extension is preferably asfurther described herein, and again such a Nanobody can also for examplealso contain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K,S112Q and/or S112K; although usually the presence of one or twoadditional substitutions at positions 110 and/or 112 will not often notbe required when position 89 is T, in which case position 110 ispreferably T and position 112 is preferably S). Also: (i) position 41may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 41 and may in particular be (or be chosen from)proline (P), serine (S), threonine (T), alanine (A) or leucine (L),which are some of the amino acid residues that most frequently occur atthis position in either humans or llamas, and may more in particular beeither proline (P) or alanine (A); and/or (ii) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (iii) position 87 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that has at least 80%, preferably at least 85%, more preferablyat least 90% such as at least 95% sequence identity with at least one ofAlb-1 (SEQ ID NO: 52 of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein)and/or Alb-23 (SEQ ID NO:61 herein) (taking into account both theframework sequences and the CDR's, but not any C-terminal extension), inwhich the amino acid residue at position 89 is T, and that optionallycontains at its C-terminal end a C-terminal extension (X)_(n) in which nis 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1or 2, such as 1); and each X is an (preferably naturally occurring)amino acid residue that is independently chosen, and preferablyindependently chosen from the group consisting of alanine (A), glycine(G), valine (V), leucine (L) or isoleucine (I) (again, such a C-terminalextension is preferably as further described herein, and again such aNanobody can also for example also contain one or more of the otherspecific amino acid residues/substitutions mentioned herein, such as11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q,T110Q, 112Q, 112K, S112Q and/or S112K; although usually the presence ofone or two additional substitutions at positions 110 and/or 112 will notoften not be required when position 89 is T, in which case position 110is preferably T and position 112 is preferably S). Also: (i) position 41may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 41 and may in particular be (or be chosen from)proline (P), serine (S), threonine (T), alanine (A) or leucine (L),which are some of the amino acid residues that most frequently occur atthis position in either humans or llamas, and may more in particular beeither proline (P) or alanine (A); and/or (ii) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (iii) position 87 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).

According to one specific aspect, any such serum albumin-bindingNanobody of the invention with a T at position 89 can also have theamino acid residues that are characteristic of Alb-23 and its variantsas described in WO 12/175400 (i.e. the amino acid motif GP on positions44 and 45, the amino acid motif SKN on positions 74 to 76, andpreferably a G at position 16 and optionally also an R at position 83).

As with the VH domains of the invention that contain an Q or K atposition 112, the VH domains of the invention that contain a T atposition 89 (optionally together with a Q or K at position 112 and/ortogether with a C-terminal extension) show reduced binding bypre-existing antibodies, and in particular by such pre-existingantibodies (as for example are found in samples obtained from SLEpatients) that are capable of binding to VH domains and Nanobodies inthe presence of a C-terminal extension.

In yet another aspect, the invention relates to an immunoglobulinheavy-chain variable domain (VH domain) in which the amino acid residueat position 89 (Kabat numbering) is leucine (L) and the amino acidresidue at position 110 is either a lysine (K) residue or glutamine (Q)residue. Such immunoglobulin heavy-chain variable domains (VH domain)with an L at position 89 and a K or Q at position 110 are also includedin the term “VH domains of the invention” as used herein in its broadestsense and can further be as described herein for the other VH domains ofthe invention (i.e. those that comprise a K or Q at position 112 or thatcomprise a T at position 89). Accordingly, such an immunoglobulinheavy-chain variable domain (VH domain) can have a C-terminal extensionas further described herein (including the indicated preferences forsuch a C-terminal extension); can be an ISVD and in particular aNanobody as further described herein.

Again, if such a VH domain of the invention has an exposed C-terminalregion (for example, because it forms the C-terminal end of the protein,polypeptide or other construct in which it is present), it preferablycontains a C-terminal extension (reference is made to the data shown inTable C below).

Also, Nanobodies of the invention in which position 89 is L and a K or Qat position 110 (i.e. with or without a C-terminal extension) preferablycontain an amino acid at position 11 that is chosen from L (the mostoften occurring amino acid residue in VHH's), E, K, M, S, V, W or Y;more preferably from L, E, K, V or Y, and even more preferably from L, Kor V (with V being most preferred). For example, they can contain anL11K or L11V substitution, as well as for example a P14A or A14Psubstitution and/or a Q108L substitution (they can also suitably containan S112K and/or S112Q mutation, although usually the presence of anadditional substitution at positions 112 will not often not be requiredwhen position 89 is L and position 110 is K or Q, in which case position112 is preferably S). Also: (i) position 41 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

Also, in one aspect, in such VH domains of the invention in which 89 isL and 110 is K or Q, the amino acid residue at position 112 is serine(S). More in particular, the C-terminal end of such a VH domain may beone of (and preferably is one of) VKVSS (SEQ ID NO: 95), VQVSS (SEQ IDNO: 96), VKVSS(X)_(n) (SEQ ID NO:97) or VQVSS(X)_(n) (SEQ ID NO: 98), inwhich n and X are as further described herein for the VH domains of theinvention in which position 112 is Q or K (and in which any C-terminalextension is preferably as further described herein for the VH domainsof the invention in which position 112 is Q or K). Also, as is the casefor the VH domains of the invention in which position 112 is Q or K orposition 89 is T, when such a VH domain with an L at position 89 and a Kor Q at position 110 is a nanobody, position 11 is preferably a leucine(L), position 14 can in particular be alanine (A) or proline (P) andposition 108 can in particular be Q or L (and in humanized nanobodiespreferably L); and such a Nanobody with an L at position 89 and a K or Qat position 110 can contain one or more nanobody hallmark residuesand/or can be suitably humanized. Also: (i) position 41 may for examplebe one of the amino acid residues (i.e. human VH3 residues and/orCamelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

Such a VH domain of the invention with an L at position 89 and a K or Qat position 110 can again also be a serum albumin Nanobody as furtherdescribed herein. For example, such a serum albumin-binding Nanobody canbe one of the sequences of SEQ ID NOs: 46 to 75, but with an L atposition 89 and a K or Q at position 110; or another serum albuminbinding Nanobody with an L at position 89 and a K or Q at position 110that has at least 80%, preferably at least 85%, more preferably at least90% such as at least 95% sequence identity with at least one of Alb-1(SEQ ID NO: 52 of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein) and/orAlb-23 (SEQ ID NO:61 herein) (taking into account both the frameworksequences and the CDR's, but not any C-terminal extension).

More generally, a VH domain against serum albumin according to thisaspect of the invention can be one of the Nanobodies against (human)serum albumin that are described in WO 2004/041865, and in particular inWO 2006/122787 and WO 2012/175400 (all applications fromapplicant/assignee), in which the amino acid at position 89 is a leucine(L) and the amino acid residue at position 110 is of K or Q, and whichis optionally provided with a C-terminal extension as described herein(and can also for example also contain one or more of the other specificamino acid residues/substitutions mentioned herein, such as 11L, L11V,L11K, 14A, P14A, 14P, A14P, 108L and/or Q108L; as well as S112K orS112Q, although usually the presence of an additional substitution atposition 112 will not often not be required when position 89 is L andposition 110 is K or Q, in which case position 112 is preferably S).Furthermore, it is envisaged that the present invention can also beapplied other serum-albumin binding heavy-chain ISVD's, such as thosedescribed in WO 03/035694, WO 04/003019, WO 05/118642, WO 06/059106, WO08/096158, WO 09/121804WO 10/108937 or US 2013/0129727, i.e. by suitablyintroducing leucine (L) at position 89 and either K or Q at position110, and optionally one or more of the other amino acidresidues/substitutions described herein) and optionally (and usuallypreferably as outlined herein) adding a C-terminal extension (as furtherdescribed herein). In these VH domains against serum albumin, the aminoacid at position 112 is preferably S, and said VH domains preferablyhave a C-terminal end that is one of SEQ ID NO's: 95 to 98. Also: (i)position 41 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 41 and may in particular be (or be chosenfrom) proline (P), serine (S), threonine (T), alanine (A) or leucine(L), which are some of the amino acid residues that most frequentlyoccur at this position in either humans or llamas, and may more inparticular be either proline (P) or alanine (A); and/or (ii) position 42may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 42 and may in particular be (or be chosen from)glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

Some preferred but non-limiting examples of such serum albumin bindingNanobodies of the invention are humanized variants of the amino acidsequence of SEQ ID NO: 52 of WO 2006/122787 (called “Alb-1” in WO2006/122787), in which the amino acid at position 89 is L and the aminoacid at position 110 is K or Q (and which is optionally provided with aC-terminal extension as described herein), such as the humanizedvariants of Alb-1 that are given in SEQ ID NO's: 57 to 64 of WO2006/122787 (in each case, with a V89L substitution and a T110Q or T110Ksubstitution, and optionally with a C-terminal extension) or thehumanized variants of Alb-1 that are given in SEQ ID NO's 3 to 11 of WO2012/175400 (again, in each case, with a V89L substitution and a T110Qor T110K substitution), of which SEQ ID NO's 3, 4 and 5 can optionallycontain a C-terminal extension, and SEQ ID NO's 6 to 11 already containa C-terminal extension (and again, such variants can contain one or moreof the other specific amino acid residues/substitutions mentionedherein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and/orQ108L; as well as S112K or S112Q, although usually the presence of anadditional substitution at position 112 will not often not be requiredwhen position 89 is L and position 110 is K or Q, in which case position112 is preferably S). In these VH domains against serum albumin, theamino acid at position 112 is preferably S, and said VH domainspreferably have a C-terminal end that is one of SEQ ID NO's: 95 to 98.Also: (i) position 41 may for example be one of the amino acid residues(i.e. human VH3 residues and/or Camelid VHH residues) mentioned in TableA-6 of WO 08/020079 for position 41 and may in particular be (or bechosen from) proline (P), serine (S), threonine (T), alanine (A) orleucine (L), which are some of the amino acid residues that mostfrequently occur at this position in either humans or llamas, and maymore in particular be either proline (P) or alanine (A); and/or (ii)position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

Thus, in a further aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

the amino acid residue at position 89 is L;

the amino acid residue at position 110 is K or Q;

and that optionally contains at its C-terminal end a C-terminalextension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such as 1,2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (again, such a C-terminal extension is preferably asfurther described herein, and again such a Nanobody can also for examplealso contain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L and/or Q108L; as well as S112K or S112Q, althoughusually the presence of an additional substitution at position 112 willnot often not be required when position 89 is L and position 110 is K orQ, in which case position 112 is preferably S). In these VH domainsagainst serum albumin, the amino acid at position 112 is preferably S,and said VH domains preferably have a C-terminal end that is one of SEQID NO's: 95 to 98. Also: (i) position 41 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that is a humanized variant of SEQ ID NO: 52 of WO 2006/122787in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

the amino acid residue at position 89 is L;

the amino acid residue at position 110 is K or Q;

and that optionally contains at its C-terminal end a C-terminalextension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such as 1,2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (again, such a C-terminal extension is preferably asfurther described herein, and again such a Nanobody can also for examplealso contain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L and/or Q108L; as well as S112K or S112Q, althoughusually the presence of an additional substitution at position 112 willnot often not be required when position 89 is L and position 110 is K orQ, in which case position 112 is preferably S)). In these VH domainsagainst serum albumin, the amino acid at position 112 is preferably S,and said VH domains preferably have a C-terminal end that is one of SEQID NO's: 95 to 98. Also: (i) position 41 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that has at least 80%, preferably at least 85%, more preferablyat least 90% such as at least 95% sequence identity with at least one ofAlb-1 (SEQ ID NO: 52 of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein)and/or Alb-23 (SEQ ID NO:61 herein) (taking into account both theframework sequences and the CDR's, but not any C-terminal extension), inwhich the amino acid residue at position 89 is L and the amino acidresidue at position 110 is K or Q, and that optionally contains at itsC-terminal end a C-terminal extension (X)_(n) in which n is 1 to 10,preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, suchas 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen, and preferably independentlychosen from the group consisting of alanine (A), glycine (G), valine(V), leucine (L) or isoleucine (I) (again, such a C-terminal extensionis preferably as further described herein, and again such a Nanobody canalso for example also contain one or more of the other specific aminoacid residues/substitutions mentioned herein, such as 11L, L11V, L11K,14A, P14A, 14P, A14P, 108L and/or Q108L; as well as S112K or S112Q,although usually the presence of an additional substitution at position112 will not often not be required when position 89 is L and position110 is K or Q, in which case position 112 is preferably S). Such aNanobody of the invention is again preferably a humanized variant ofAlb-1 (but with a V89L and T110K or T110Q substitution), and morepreferably has at least one, in particular any two, and more inparticular all three of CDR1, CDR2 and/or CDR3 given in SEQ ID NO's 41to 43, respectively. In these VH domains against serum albumin, theamino acid at position 112 is preferably S, and said VH domainspreferably have a C-terminal end that is one of SEQ ID NO's: 95 to 98.Also: (i) position 41 may for example be one of the amino acid residues(i.e. human VH3 residues and/or Camelid VHH residues) mentioned in TableA-6 of WO 08/020079 for position 41 and may in particular be (or bechosen from) proline (P), serine (S), threonine (T), alanine (A) orleucine (L), which are some of the amino acid residues that mostfrequently occur at this position in either humans or llamas, and maymore in particular be either proline (P) or alanine (A); and/or (ii)position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

According to one specific aspect, any such serum albumin-bindingNanobody of the invention with an L at position 89 and a K or Q atposition 110 can also have the amino acid residues that arecharacteristic of Alb-23 and its variants as described in WO 12/175400(i.e. the amino acid motif GP on positions 44 and 45, the amino acidmotif SKN on positions 74 to 76, and preferably a G at position 16 andoptionally also an R at position 83). Also: (i) position 41 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

As with the VH domains of the invention that contain an Q or K atposition 112 or that have a T at position 89, the VH domains of theinvention that contain an L at position 89 and a K or Q at position 110show reduced binding by pre-existing antibodies, and in particular bysuch pre-existing antibodies (as for example are found in samplesobtained from SLE patients) that are capable of binding to VH domainsand Nanobodies in the presence of a C-terminal extension.

Some non-limiting examples of VH domains of the invention that have an Lat position 89 and a K at position 110 (and in addition, a V at position11) are given in FIG. 2 as SEQ ID NO's: 123-136. These are VH domainsbinding to human serum albumin and have the CDR's indicated herein forthe preferred serum albumin binding VH domains of the invention.

In yet another aspect, the invention relates to an immunoglobulinheavy-chain variable domain (VH domain) in which the amino acid residueat position 89 (Kabat numbering) is leucine (L) and the amino acidresidue at position 11 is valine (V). Such immunoglobulin heavy-chainvariable domains (VH domain) with an L at position 89 and a V atposition 11 are also included in the term “VH domains of the invention”as used herein in its broadest sense and can further be as describedherein for the other VH domains of the invention described herein (i.e.those that comprise a K or Q at position 112, that comprise a T atposition 89, or that comprise an L at position 89 and a K or Q atposition 110; albeit that for the VH domains according to the currentaspect, the amino acid residue at position 89 will be L, at amino acidresidue at position 11 will be V, and the amino acid residues atpositions 110 and 112, respectively, can be any amino acid residuesuitable for these positions). Accordingly, such an immunoglobulinheavy-chain variable domain (VH domain) can have a C-terminal extensionas further described herein (including the indicated preferences forsuch a C-terminal extension); can be an ISVD and in particular aNanobody as further described herein.

Again, if such a VH domain of the invention has an exposed C-terminalregion (for example, because it forms the C-terminal end of the protein,polypeptide or other construct in which it is present), it preferablycontains a C-terminal extension (reference is made to the data shown inTable C below).

Also, Nanobodies of the invention in which position 89 is L and position11 is V (i.e. with or without a C-terminal extension): (i) preferablycontain an amino acid at position 110 that is chosen from T, I, A, K orQ (and preferably from T, K or Q, and that in particular can be T); (ii)preferably contain an amino acid at position 112 that is chosen from S,F, K or Q (and more preferably S, K or Q, and that in particular can beS); and (iii) can for example also contain a P14A or A14P substitutionand/or a Q108L substitution. According to one specific embodiment, inthe VH domains according to this aspect of the invention, the amino acidresidue at position 110 is T and the amino acid residue at position 112is S, and more preferably the C-terminal end is either VTVSS (SEQ ID NO:77) or VTVSS(X)n (SEQ ID NO: 78), in which X and n are as defined hereinfor the C-terminal extension of the other VH domains of the invention.Also, in the VH domains according to this aspect of the invention: (i)position 41 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 41 and may in particular be (or be chosenfrom) proline (P), serine (S), threonine (T), alanine (A) or leucine(L), which are some of the amino acid residues that most frequentlyoccur at this position in either humans or llamas, and may more inparticular be either proline (P) or alanine (A); and/or (ii) position 42may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 42 and may in particular be (or be chosen from)glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

Thus, for example, in such VH domains of the invention in which 89 is Land 11 is V, the C-terminal end of such a VH domain may be one of (andpreferably is one of) VTVSS (SEQ ID NO:77), VTVSS(X)n (SEQ ID NO:78),VTVKS (SEQ ID NO:1), VTVKS(X)n (SEQ ID NO:21), VTVQS (SEQ ID NO:2),VTVQS(X)n (SEQ ID NO:22), VKVSS (SEQ ID NO: 95), VKVSS(X)n (SEQ IDNO:97), VQVSS (SEQ ID NO: 96), VQVSS(X)n (SEQ ID NO: 98), VZVZS (SEQ IDNO: 107, in which each amino acid residue Z is independently K or Q) orVZVZSX(n) (SEQ ID NO:108, in which each amino acid residue Z isindependently K or Q) (and is in particular one of VTVSS, VKVSS (SEQ IDNO: 95), VQVSS (SEQ ID NO: 96), VKVSS(X)_(n) (SEQ ID NO:97) orVQVSS(X)_(n) (SEQ ID NO: 98), and may more in particular be either VTVSS(SEQ ID NO:77) or VTVSS(X)n (SEQ ID NO:78)), in which n and X are asfurther described herein for the VH domains of the invention in whichposition 112 is Q or K (and in which any C-terminal extension ispreferably as further described herein for the VH domains of theinvention in which position 112 is Q or K). Also, as is the case for theVH domains of the invention in which position 112 is Q or K or position89 is T, when such a VH domain with an L at position 89 and a V atposition 11 is a nanobody, position 14 can in particular be alanine (A)or proline (P) and position 108 can be Q or L (and in humanizednanobodies preferably L); and such a Nanobody with an L at position 89and a V at position 11 can contain one or more nanobody hallmarkresidues and/or can be suitably humanized. Also: (i) position 41 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

Such a VH domain of the invention with an L at position 89 and a V atposition 11 can again also be a serum albumin Nanobody as furtherdescribed herein. For example, such a serum albumin-binding Nanobody canbe one of the sequences of SEQ ID NOs: 46 to 75, but with an L atposition 89 and a V at position 11; or another serum albumin bindingNanobody with an L at position 89 and a V at position 11 that has atleast 80%, preferably at least 85%, more preferably at least 90% such asat least 95% sequence identity with at least one of Alb-1 (SEQ ID NO: 52of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein) and/or Alb-23 (SEQ IDNO:61 herein) (taking into account both the framework sequences and theCDR's, but not any C-terminal extension).

More generally, a VH domain against serum albumin according to thisaspect of the invention can be one of the Nanobodies against (human)serum albumin that are described in WO 2004/041865, and in particular inWO 2006/122787 and WO 2012/175400 (all applications fromapplicant/assignee), in which the amino acid at position 89 is a leucine(L) and the amino acid residue at position 11 is V, and which isoptionally provided with a C-terminal extension as described herein (andcan also for example also suitably contain one or more of the otherspecific amino acid residues/substitutions mentioned herein, such as14A, P14A, 14P, A14P, 108L, Q108L, V110K, V110Q, S112K and/or S112Q).Furthermore, it is envisaged that the present invention can also beapplied other serum-albumin binding heavy-chain ISVD's, such as thosedescribed in WO 03/035694, WO 04/003019, WO 05/118642, WO 06/059106, WO08/096158, WO 09/121804WO 10/108937 or US 2013/0129727, i.e. by suitablyintroducing leucine (L) at position 89 and valine at position 11, andoptionally one or more of the other amino acid residues/substitutionsdescribed herein) and optionally (and usually preferably as outlinedherein) adding a C-terminal extension (as further described herein). Inthese VH domains against serum albumin, the amino acid at position 112is preferably S, and said VH domains preferably have a C-terminal endthat is one of SEQ ID NO's: 95 to 98. Also: (i) position 41 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

Some preferred but non-limiting examples of such serum albumin bindingNanobodies of the invention are humanized variants of the amino acidsequence of SEQ ID NO: 52 of WO 2006/122787 (called “Alb-1” in WO2006/122787), in which the amino acid at position 89 is L and the aminoacid at position 11 is V (and which is optionally provided with aC-terminal extension as described herein), such as the humanizedvariants of Alb-1 that are given in SEQ ID NO's: 57 to 64 of WO2006/122787 (in each case, with a V89L substitution and a L11Vsubstitution, and optionally with a C-terminal extension) or thehumanized variants of Alb-1 that are given in SEQ ID NO's 3 to 11 of WO2012/175400 (again, in each case, with a V89L substitution and a Lily),of which SEQ ID NO's 3, 4 and 5 can optionally contain a C-terminalextension, and SEQ ID NO's 6 to 11 already contain a C-terminalextension (and again, such variants can suitably contain one or more ofthe other specific amino acid residues/substitutions mentioned herein,such as 14A, P14A, 14P, A14P, 108L, Q108L, T110K, T110Q, S112K and/orS112Q). In these VH domains against serum albumin with an L at position89 and a V at position 11, the amino acid at position 110 is preferablyT and the amino acid residue at position 112 is preferably S, and saidVH domains preferably have a C-terminal end that is one of SEQ ID NO's:95 to 98. Also: (i) position 41 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 41 and may in particular be(or be chosen from) proline (P), serine (S), threonine (T), alanine (A)or leucine (L), which are some of the amino acid residues that mostfrequently occur at this position in either humans or llamas, and maymore in particular be either proline (P) or alanine (A); and/or (ii)position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (iii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A).

Thus, in a further aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

the amino acid residue at position 89 is L;

the amino acid residue at position 11 is V;

in which preferably

the amino acid residue at position 110 is one of K, Q or T, and morepreferably T;

the amino acid residue at position 112 is one of K, Q or S, and morepreferably S; and that optionally contains at its C-terminal end aC-terminal extension (X)_(n) in which n is 1 to 10, preferably 1 to 5,such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each Xis an (preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (again, such a C-terminal extension is preferably asfurther described herein, and again such a Nanobody can also for examplealso suitably contain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 14A, P14A, 14P, A14P,108L, Q108L). Again, in these VH domains against serum albumin with an Lat position 89 and a V at position 11, the amino acid at position 110 ispreferably T and the amino acid at position 112 is preferably S, andsaid VH domains preferably have a C-terminal end that is one of SEQ IDNO's: 95 to 98. Also: (i) position 41 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that is a humanized variant of SEQ ID NO: 52 of WO 2006/122787in which:

CDR1 is the amino acid sequence (SEQ ID NO: 41) SFGMS;CDR2 is the amino acid sequence (SEQ ID NO: 42) SISGSGSDTLYADSVKG;CDR3 is the amino acid sequence (SEQ ID NO: 43) GGSLSR;

the amino acid residue at position 89 is L;

the amino acid residue at position 11 is V;

and preferably:

the amino acid residue at position 110 is one of K, Q or T, and morepreferably T;

the amino acid residue at position 112 is one of K, Q or S, and morepreferably S;

and that optionally contains at its C-terminal end a C-terminalextension (X)_(n) in which n is 1 to 10, preferably 1 to 5, such as 1,2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (again, such a C-terminal extension is preferably asfurther described herein, and again such a Nanobody can also for examplealso contain one or more of the other specific amino acidresidues/substitutions mentioned herein, such as 11L, L11V, L11K, 14A,P14A, 14P, A14P, 108L and/or Q108L). In these VH domains against serumalbumin with an L at position 89 and a V at position 11, the amino acidresidue at position 110 is preferably T and the amino acid residue atposition 112 is preferably S, and said VH domains preferably have aC-terminal end that is one of SEQ ID NO's: 95 to 98. Also: (i) position41 may for example be one of the amino acid residues (i.e. human VH3residues and/or Camelid VHH residues) mentioned in Table A-6 of WO08/020079 for position 41 and may in particular be (or be chosen from)proline (P), serine (S), threonine (T), alanine (A) or leucine (L),which are some of the amino acid residues that most frequently occur atthis position in either humans or llamas, and may more in particular beeither proline (P) or alanine (A); and/or (ii) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (iii) position 87 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).

In a particular aspect, the invention relates to a Nanobody of theinvention (as defined herein) that can bind (and in particularspecifically bind) to serum albumin (and in particular human serumalbumin) that has at least 80%, preferably at least 85%, more preferablyat least 90% such as at least 95% sequence identity with at least one ofAlb-1 (SEQ ID NO: 52 of WO 2006/122787), Alb-8 (SEQ ID NO: 46 herein)and/or Alb-23 (SEQ ID NO:61 herein) (taking into account both theframework sequences and the CDR's, but not any C-terminal extension), inwhich the amino acid residue at position 89 is L and the amino acidresidue at position 11 is V, and that optionally contains at itsC-terminal end a C-terminal extension (X)_(n) in which n is 1 to 10,preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, suchas 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen, and preferably independentlychosen from the group consisting of alanine (A), glycine (G), valine(V), leucine (L) or isoleucine (I) (again, such a C-terminal extensionis preferably as further described herein, and again such a Nanobody canalso for example also suitably contain one or more of the other specificamino acid residues/substitutions mentioned herein, such as 14A, P14A,14P, A14P, 108L, Q108L, T110K, T110Q, S112K and/or S112Q). Such aNanobody of the invention is again preferably a humanized variant ofAlb-1 (but with a V89L and L11V substitution), and more preferably hasat least one, in particular any two, and more in particular all three ofCDR1, CDR2 and/or CDR3 given in SEQ ID NO's 41 to 43, respectively. Inthese VH domains against serum albumin, the amino acid at position 112is preferably S, and said VH domains preferably have a C-terminal endthat is one of SEQ ID NO's: 95 to 98. Also: (i) position 41 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

According to one specific aspect, any such serum albumin-bindingNanobody of the invention with an L at position 89 and a V at position11 can also have the amino acid residues that are characteristic ofAlb-23 and its variants as described in WO 12/175400 (i.e. the aminoacid motif GP on positions 44 and 45, the amino acid motif SKN onpositions 74 to 76, and preferably a G at position 16 and optionallyalso an R at position 83). Also: (i) position 41 may for example be oneof the amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 41 and mayin particular be (or be chosen from) proline (P), serine (S), threonine(T), alanine (A) or leucine (L), which are some of the amino acidresidues that most frequently occur at this position in either humans orllamas, and may more in particular be either proline (P) or alanine (A);and/or (ii) position 42 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-6 of WO 08/020079 for position 42 and may in particular be(or be chosen from) glycine (G) or glutamic acid (E); and/or (iii)position 87 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-7of WO 08/020079 for position 87 and may in particular be (or be chosenfrom) threonine (T) or alanine (A).

As with the VH domains of the invention that contain an Q or K atposition 112 or that have a T at position 89, the VH domains of theinvention that contain an L at position 89 and a V at position 11 showreduced binding by pre-existing antibodies, and in particular by suchpre-existing antibodies (as for example are found in samples obtainedfrom SLE patients) that are capable of binding to VH domains andNanobodies in the presence of a C-terminal extension. One further thingto note with the VH domains of the invention is which position 89 is Land position 11 is V is that these substitutions are known to occur withsome frequency in human VH domains (see Table A-5 of WO 08/020079 forposition 11 and Table A-7 of WO 08/020079 of position 89).

Some non-limiting examples of VH domains of the invention that have a Vat position 11 and a Tat position 89 are given in FIG. 2 as SEQ ID NO's:109-136. Of these VH domains, the sequences given as SEQ ID NO's:123-136 contain, in addition to the L11V and V89L mutations, also aT110K mutation. These are VH domains binding to human serum albumin andhave the CDR's indicated herein for the preferred serum albumin bindingVH domains of the invention.

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is V; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

and in which said VH domain optionally contains a C-terminal extension(X)_(n), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or5 (and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Preferably, in the VH domains according to thisparagraph, the amino acid residue at position 110 is T and the aminoacid residue at position 112 is S. Again, as with the other VH domainsof the invention, such a VH domain may be directed against any suitabletarget (and in particular a therapeutically relevant target). Accordingto one specific aspect, such a VH domain is directed to serum albumin.

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q; and/or (ii) the amino acid residue at position 89 is T; and/or(iii) the amino acid residue at position 89 is L and the amino acidresidue at position 110 is one of K or Q; and (iv) in each of cases (i)to (iii), the amino acid at position 11 is preferably V; andin which said VH domain optionally contains a C-terminal extension(X)_(n), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or5 (and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is one of T, V or L (and is preferablyV), and the amino acid residue at position 110 is one of T, K or Q (andis preferably T); and/or (ii) the amino acid residue at position 89 isT, and the amino acid residue at position 112 is one of S, K or Q (andis preferably S), and the amino acid residue at position 110 is one ofT, K or Q (and is preferably T); and/or (iii) the amino acid residue atposition 89 is L and the amino acid residue at position 110 is one of Kor Q, and the amino acid residue at position 112 is one of S, K or Q(and is preferably S); and (iv) in each of cases (i) to (iii), the aminoacid at position 11 is preferably V; and in which said VH domainoptionally contains a C-terminal extension (X)_(n), in which n is 1 to10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2,such as 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen, and preferably independentlychosen from the group consisting of alanine (A), glycine (G), valine(V), leucine (L) or isoleucine (I) (which C-terminal extension ispreferably as further described herein). Also: (i) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (ii) position 87 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).Another aspect of the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody) which is as described in this paragraph, inwhich the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q; or (ii) the amino acid residue at position 89 is T; or (iii) theamino acid residue at position 89 is L and the amino acid residue atposition 110 is one of K or Q; and (iv) in each of cases (i) to (iii),the amino acid at position 11 is preferably V; andin which said VH domain optionally contains a C-terminal extension(X)_(n), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or5 (and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is one of T, V or L (and is preferablyV), and the amino acid residue at position 110 is one of T, K or Q (andis preferably T); or (ii) the amino acid residue at position 89 is T,and the amino acid residue at position 112 is one of S, K or Q (and ispreferably S), and the amino acid residue at position 110 is one of T, Kor Q (and is preferably T); or (iii) the amino acid residue at position89 is L, and the amino acid residue at position 110 is one of K or Q,and the amino acid residue at position 112 is one of S, K or Q (and ispreferably S); and (iv) in each of cases (i) to (iii), the amino acid atposition 11 is preferably V;and in which said VH domain optionally contains a C-terminal extension(X)_(n), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or5 (and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is V, and the amino acid residue atposition 110 is T; or (ii) the amino acid residue at position 89 is T,the amino acid residue at position 112 is S, and the amino acid residueat position 110 is T; or (iii) the amino acid residue at position 89 isL and the amino acid residue at position 110 is one of K or Q, and theamino acid residue at position 112 is S (and is preferably S); and (iv)in each of cases (i) to (iii), the amino acid at position 11 ispreferably V; andin which said VH domain optionally contains a C-terminal extension(X)_(n), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or5 (and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q; and/or (ii) the amino acid residue at position 89 is T; and/or(iii) the amino acid residue at position 89 is L and the amino acidresidue at position 110 is one of K or Q; and (iv) in each of cases (i)to (iii), the amino acid at position 11 is preferably V; andin which said VH domain contains a C-terminal extension (X)_(n), inwhich n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is one of T, V or L (and is preferablyV), and the amino acid residue at position 110 is one of T, K or Q (andis preferably T); and/or (ii) the amino acid residue at position 89 isT, and the amino acid residue at position 112 is one of S, K or Q (andis preferably S), and the amino acid residue at position 110 is one ofT, K or Q (and is preferably T); and/or (iii) the amino acid residue atposition 89 is L and the amino acid residue at position 110 is one of Kor Q, and the amino acid residue at position 112 is one of S, K or Q(and is preferably S); and (iv) in each of cases (i) to (iii), the aminoacid at position 11 is preferably V; and in which said VH domaincontains a C-terminal extension (X)_(n), in which n is 1 to 10,preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, suchas 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen, and preferably independentlychosen from the group consisting of alanine (A), glycine (G), valine(V), leucine (L) or isoleucine (I) (which C-terminal extension ispreferably as further described herein). Also: (i) position 42 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 42 and may in particular be (or be chosen from) glycine (G) orglutamic acid (E); and/or (ii) position 87 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-7 of WO 08/020079 for position 87 and mayin particular be (or be chosen from) threonine (T) or alanine (A).Another aspect of the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody) which is as described in this paragraph, inwhich the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q; or (ii) the amino acid residue at position 89 is T; or (iii) theamino acid residue at position 89 is L and the amino acid residue atposition 110 is one of K or Q; and (iv) in each of cases (i) to (iii),the amino acid at position 11 is preferably V; andin which said VH domain contains a C-terminal extension (X)_(n), inwhich n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is one of T, V or L (and is preferablyV), and the amino acid residue at position 110 is one of T, K or Q (andis preferably T); or (ii) the amino acid residue at position 89 is T,and the amino acid residue at position 112 is one of S, K or Q (and ispreferably S), and the amino acid residue at position 110 is one of T, Kor Q (and is preferably T); or (iii) the amino acid residue at position89 is L, and the amino acid residue at position 110 is one of K or Q,and the amino acid residue at position 112 is one of S, K or Q (and ispreferably S); and (iv) in each of cases (i) to (iii), the amino acid atposition 11 is preferably V; and in which said VH domain contains aC-terminal extension (X)_(n), in which n is 1 to 10, preferably 1 to 5,such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each Xis an (preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I) (which C-terminal extension is preferably as furtherdescribed herein). Also: (i) position 42 may for example be one of theamino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (ii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A). Another aspect of theinvention relates to a VH domain (and in particular a VH domain that isan ISVD, and more in particular a VH domain that is a nanobody) which isas described in this paragraph, in which the amino acid residue atposition 11 is V and the amino acid residue at position 110 is either Kor Q (and in which the amino acid residues at positions 14, 41, 89, 108and 112 can be as listed in the above bullet points, the amino acidresidues at positions 42 and 87 can for example be as described in thisparagraph, and the VH domain can optionally contain a C-terminalextension (X)_(n) as described in this paragraph).

In a further aspect, the invention relates to a VH domain (and inparticular a VH domain that is an ISVD, and more in particular a VHdomain that is a nanobody), in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q, the amino at position 89 is V, and the amino acid residue atposition 110 is T; or (ii) the amino acid residue at position 89 is T,the amino acid residue at position 112 is S, and the amino acid residueat position 110 is T; or (iii) the amino acid residue at position 89 isL and the amino acid residue at position 110 is one of K or Q, and theamino acid residue at position 112 is S (and is preferably S); and (iv)in each of cases (i) to (iii), the amino acid at position 11 ispreferably V; andin which said VH domain contains a C-terminal extension (X)_(n), inwhich n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I) (whichC-terminal extension is preferably as further described herein). Also:(i) position 42 may for example be one of the amino acid residues (i.e.human VH3 residues and/or Camelid VHH residues) mentioned in Table A-6of WO 08/020079 for position 42 and may in particular be (or be chosenfrom) glycine (G) or glutamic acid (E); and/or (ii) position 87 may forexample be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-7 of WO 08/020079 forposition 87 and may in particular be (or be chosen from) threonine (T)or alanine (A). Another aspect of the invention relates to a VH domain(and in particular a VH domain that is an ISVD, and more in particular aVH domain that is a nanobody) which is as described in this paragraph,in which the amino acid residue at position 11 is V and the amino acidresidue at position 110 is either K or Q (and in which the amino acidresidues at positions 14, 41, 89, 108 and 112 can be as listed in theabove bullet points, the amino acid residues at positions 42 and 87 canfor example be as described in this paragraph, and the VH domain canoptionally contain a C-terminal extension (X)_(n) as described in thisparagraph).

Again, in the VH domains of the invention as defined herein, the aminoacid residues at positions that are not explicitly defined herein can beany amino acid residue that is suitable at such a position for VHdomains, and in particular for ISVD's and more in particular fornanobodies (including humanized VHH domains). Reference is again made tothe prior art cited herein, such as for example Tables A-3 and A-5 toA-8 of WO 08/020079. Preferably, in each case, the amino acid residue atposition 11 is L or V, and more preferably V. Also: (i) position 41 mayfor example be one of the amino acid residues (i.e. human VH3 residuesand/or Camelid VHH residues) mentioned in Table A-6 of WO 08/020079 forposition 41 and may in particular be (or be chosen from) proline (P),serine (S), threonine (T), alanine (A) or leucine (L), which are some ofthe amino acid residues that most frequently occur at this position ineither humans or llamas, and may more in particular be either proline(P) or alanine (A); and/or (ii) position 42 may for example be one ofthe amino acid residues (i.e. human VH3 residues and/or Camelid VHHresidues) mentioned in Table A-6 of WO 08/020079 for position 42 and mayin particular be (or be chosen from) glycine (G) or glutamic acid (E);and/or (iii) position 87 may for example be one of the amino acidresidues (i.e. human VH3 residues and/or Camelid VHH residues) mentionedin Table A-7 of WO 08/020079 for position 87 and may in particular be(or be chosen from) threonine (T) or alanine (A).

Also, said VH domain of the invention can be directed against anysuitable target, and in particular a therapeutic target. In one aspect,they are directed against a human serum protein such as human serumalbumin.

The invention also relates to proteins, polypeptides, constructs,compounds or other chemical entities that comprise at least one VHdomain of the invention (also collectively referred to herein as“compounds of the invention”).

As further described herein, according to one specific aspect, in acompound of the invention, the VH domain of the invention is presentat/forms the C-terminal end of the same. In such a case, the VH domainof the invention that forms/is present at the C-terminal end of thecompound of the invention preferably has a C-terminal extension asdescribed herein.

As also further described herein, the compounds of the invention can bea ScFv, diabody or another protein, polypeptide or construct in whichthe one or more VH domains of the invention are associated with one ormore VL domains to form one or more functional antigen-binding sites.

However, according to a preferred aspect of the invention, the VHdomains of the invention are ISVD's and the compounds of the inventionare proteins, polypeptides, constructs, compounds or other chemicalentities that comprise or essentially consist of at least one ISVD ofthe invention and optionally one or more further amino acid sequences,moieties, binding domains or binding units (suitably linked to eachother, optionally via one or more linkers). In particular, suchcompounds of the invention can comprise or essentially consist of one ormore ISVD's, at least one of which is an ISVD of the invention. Such acompound of the invention may in particular have an ISVD of theinvention at its C-terminal end, in which case the ISVD of the inventionalso preferably has a C-terminal extensions as described herein. Also,if such a compound of the invention contains two or more ISVDs, two ormore or essentially all of the ISVD's present may be ISVD's of theinvention (i.e. each having at least one of the following amino acidresidues/substitutions: 112K, 112Q, S112K, S112Q, 89T and/or V89T, orthe combination of V89L with T110K or T110Q; and optionally one or moreof the further substitutions mentioned herein for ISVD's of theinvention, such as L11V). Also, in such a compound of the invention, theISVD of the invention is preferably a Nanobody of the invention, and allor essentially all of the ISVD's present in the compound of theinvention may be (and preferably are) Nanobodies (and in particularNanobodies of the invention, i.e. each having at least one of thefollowing amino acid residues/substitutions: 112K, 112Q, S112K, S112Q,89T and/or V89T, or the combination of V89L with T110K or T110Q; andoptionally one or more of the further substitutions mentioned herein forNanobodies of the invention, such as L11V). Examples of such compoundsof the invention will be clear to the skilled person based on thefurther disclosure herein.

Some non-limiting examples of proteins, polypeptides, constructs,compounds or other chemical entities that comprise one or more ISVD's(including at least one ISVD of the invention) are multivalent,multispecific (such as bispecific) or multiparatopic (such asbiparatopic) constructs that contain two or more ISVD's linked directlyor via one or more suitable linkers. Again, the ISVD's are preferablyNanobodies. For some non-limiting examples of such constructs and ageneral teaching on how such constructs can be made (in particular basedon Nanobodies) reference is for example made to Conrath et al., JBC 276,10(9), 7346 (2001) as well as to the review article by Muyldermans.Reviews in Mol. Biotechnol., 74: 27 (2001).

For example, such a compound of the invention containing two or moreISVD's (at least one of which is an ISVD of the invention) may be abivalent, trivalent, tetravalent or pentavalent construct, and/or may bea monospecific, bispecific, trispecific construct, and/or may be abiparatopic or triparatopic construct. Reference is again made to theprior art on IVSD-based and Nanobody-based biologicals cited herein.Also, such a compound of the invention may have been provided with anincreased half-life by functionalization and/or by including in theconstruct a moiety or binding unit that increases the half-life of theconstruct. Examples of such functionalization, moieties or binding unitswill be clear to the skilled person and may for example be as describedherein, and for example may include pegylation, fusion to serum albumin,or fusion to a peptide or binding unit that can bind to a serum proteinsuch as serum albumin. Such a serum-albumin binding peptide or bindingdomain may be any suitable serum-albumin binding peptide or bindingdomain capable of increasing the half-life of the construct (compared tothe same construct without the serum-albumin binding peptide or bindingdomain), and may in particular be serum albumin binding peptides asdescribed in WO 2008/068280 by applicant (and in particular WO2009/127691 and the non-prepublished U.S. application 61/301,819, bothby applicant), or a serum-albumin binding ISV (such as a serum-albuminbinding Nanobody; for example Alb-1 or a humanized version of Alb-1 suchas Alb-8, for which reference is for example made to WO 06/122787), oran ISVD of the invention that is directed against a (human) serumprotein such as (human) serum albumin (as further described herein).Generally, any compound of the invention with increased half-life willpreferably have a half-life (as defined herein) in human subjects of atleast 1 day, preferably at least 3 days, more preferably at least 7days, such as at least 10 days.

When the compound of the invention comprises at least one (andpreferably one) ISVD of the invention (and in particular Nanobody of theinvention) that is directed against a (human) serum protein and inparticular against (human) serum albumin, the compound of the inventionwill usually further contain one or more other therapeutically activeamino acid sequences, moieties, binding domains or binding units (i.e.directed against a therapeutically relevant target, pathway ormechanism), and the ISVD of the invention will function to extend thehalf-life of the same (and of the entire compound). Again, said one ormore further therapeutically active moieties are preferably ISVD's (andmore preferably Nanobodies), and may also be IVSD's of the invention(and more preferably Nanobodies of the invention). In such compounds ofthe invention, the ISVD of the invention that is directed against humanserum albumin may again be present at/forms the C-terminal end of thecompound, and in that case may (and preferably does) comprise aC-terminal extension as described herein. When a compound of theinvention contains an ISVD of the invention that is directed against(human) serum albumin, said the compound of the invention preferably hasa half-life (as defined herein) of at least 1 day, preferably at least 3days, more preferably at least 7 days, such as at least 10 days in thehuman subject to which the same is/has been administered. Somenon-limiting examples of ISVD's of the invention against human serumalbumin that can be used for this purpose are as further describedherein.

In one aspect, all of the ISVD's or nanobodies present in said compoundof the invention are ISVD's of the invention (meaning that they have theamino acid residues/substitutions that are characteristic of the VHdomains of the invention as defined herein, i.e. at least 112K or Q, orat least 89T, or at least 89L in combination with 110K or 110Q). Whenall the ISVD's in the compound of the invention are ISVD's of theinvention, they can have the same substitutions (for example all have anS112K or S112Q substitution) or different substitutions (for example,one can have an S1112K or S112Q substitution, and another can have aV89L mutation in combination with T110K or T110Q). Also, usually onlythe ISVD at the C-terminal end of the compound of the invention willhave a C-terminal extension (as the others will likely be linked attheir C-terminal end to another ISVD present in the compound).

Thus, in a further aspect, the invention relates a protein, polypeptideor other compound or molecule that comprises or essentially consists ofan ISVD of the invention (as further described herein).

The invention further relates to a protein, polypeptide or othercompound or molecule that comprises at least one ISVD of the inventionand at least one other therapeutic moiety or entity (either linkeddirectly or via a suitable linker).

The invention further relates to a protein, polypeptide or othercompound or molecule that comprises at least one ISVD of the inventionthat is directed against a (human) serum protein (and preferably againsthuman serum albumin) and at least one other therapeutic moiety or entity(either linked directly or via a suitable linker).

The invention further relates to a protein, polypeptide or othercompound or molecule that comprises at least two (such as two, three orfour) immunoglobulin single variable domains (either linked directly orvia a suitable linker), at least one of which is an ISVD of theinvention. In this aspect: (i) the ISVD's present may suitably be thesame or different; and when they are different they may be directedagainst the same target (for example, they may have different sequencesand/or be directed against different epitopes on the same target) oragainst two or more different targets (i.e. such that the resultingprotein, polypeptide or other compound or molecule is a bi- ormultispecific construct); and/or (ii) the ISVD present at the C-terminalend of the protein, polypeptide or other compound or molecule may or maynot be an ISVD of the invention (but preferably is); and/or (iii) whenan ISVD of the invention is present at the C-terminal end of theprotein, polypeptide or other compound or molecule, it preferably has aC-terminal extension as described herein; and/or (iv) essentially all ofthe ISVD's present in the protein, polypeptide or other compound ormolecule may be ISVD's of the invention. Also, when the ISVD's aredirected against different targets (as least one of which is atherapeutic target), according to one further aspect at least one of theISVD's present may be directed against a (human) serum protein such ashuman serum albumin (and this ISVD may or may not be an ISVD of theinvention; and when it is an ISVD of the invention, it is preferably ananobody against human serum albumin that is as further describedherein).

The invention further relates to such a protein, polypeptide or othercompound or molecule that comprises or essentially consists of twoimmunoglobulin single variable domains (either linked directly or via asuitable linker).

The invention further relates to such a protein, polypeptide or othercompound or molecule that comprises or essentially consists of threeimmunoglobulin single variable domains (either linked directly or via asuitable linker).

The invention further relates to such a protein, polypeptide or othercompound or molecule comprises or essentially consists of fourimmunoglobulin single variable domains (either linked directly or via asuitable linker).

The invention further relates to such a protein, polypeptide or othercompound or molecule that further comprises at least one moiety, bindingdomain or binding unit that confers an increased half-life to saidprotein, polypeptide or other compound or molecule (i.e. compared to thecorresponding protein, polypeptide or other compound or molecule withoutsaid moiety, binding domain or binding unit). According to a morespecific aspect, said at least one moiety, binding domain or bindingunit that confers an increased half-life to said protein, polypeptide orother compound or molecule is an immunoglobulin single variable domain,more in particular an immunoglobulin single variable domain that isdirected against a serum protein (such as serum albumin), and inparticular against a human serum protein (such as human serum albumin);and as described herein may in particular be an ISVD of the invention.Said ISVD against the serum protein may be at the N-terminal end of theprotein, polypeptide or other compound or molecule, at the C-terminalend, or (if the protein, polypeptide or other compound or moleculecomprises more than two ISVD's) in the middle of the molecule.

The invention further relates to such a protein, polypeptide or othercompound or molecule comprises or essentially consists of either:

-   -   two immunoglobulin single variable domains (either linked        directly or via a suitable linker), i.e. (said) one        immunoglobulin single variable domain (such as a nanobody) that        confers an increased half-life and one other immunoglobulin        single variable domain (such as a nanobody) that may in        particular be directed against a therapeutic target;    -   three immunoglobulin single variable domains (either linked        directly or via a suitable linker), i.e. (said) one        immunoglobulin single variable domain (such as a nanobody) that        confers an increased half-life and two other immunoglobulin        single variable domains (such as two other nanobodies) that may        in particular be directed against a therapeutic target (in which        said two other immunoglobulin single variable domains may be        directed against the same target, against two different targets        or against two different epitopes on the same target); or    -   four immunoglobulin single variable domains (either linked        directly or via a suitable linker), i.e. (said) one        immunoglobulin single variable domain (such as a nanobody) that        confers an increased half-life and two other immunoglobulin        single variable domains (such as two other nanobodies) that may        in particular be directed against a therapeutic target (in which        said three other immunoglobulin single variable domains may be        directed against the same target, against two or three different        targets and/or against two or three different epitopes on the        same target).

Again, in such a protein, polypeptide or other compound or molecule: (i)the ISVD's present may suitably be the same or different; and when theyare different they may be directed against the same target (for example,they may have different sequences and/or be directed against differentepitopes on the same target) or against two or more different targets(i.e. such that the resulting protein, polypeptide or other compound ormolecule is a bi- or multispecific construct); and/or (ii) the ISVDpresent at the C-terminal end of the protein, polypeptide or othercompound or molecule may or may not be an ISVD of the invention (butpreferably is); and/or (iii) when an ISVD of the invention is present atthe C-terminal end of the protein, polypeptide or other compound ormolecule, it preferably has a C-terminal extension as described herein;and/or (iv) essentially all of the ISVD's present in the protein,polypeptide or other compound or molecule may be ISVD's of theinvention.

The invention further relates to methods forexpressing/producing/manufacturing the VH domains of the invention andthe compounds of the invention (as further described herein). Forexample, a VH domain of the invention can be expressed/produced bysuitably expressing a nucleic acid that encodes the same in a suitablehost organism. Reference is for example made to WO 08/020079 (as well asto some of the other patent applications of applicant/assignee citedherein), that generally describes suitable methods and techniques forexpressing/producing Nanobodies, which methods can also suitably be usedto express/produce Nanobodies of the invention. Methods for expressingVH domains of the invention other than nanobodies will also be clear tothe skilled person based on the disclosure and prior art cited herein.Compounds of the invention can be suitably manufactured/produced bysuitably linking (usually via covalent bonds) one or more VH domains ofthe invention to the one or more further amino acid residues (and/orother groups or moieties) that are to be present in the final compoundof the invention, optionally via one or more linkers or spacers.Alternatively, when a compound of the invention is a protein orpolypeptide, it can be manufactured/produced by suitably expressing anucleic acid that encodes the same in a suitable host organism.Reference is again for example made to the general methods described inWO 08/020079 and in some of the other patent applications ofapplicant/assignee cited herein.

The invention also relates to a nucleotide sequence and/or nucleic acidthat encodes a VH domain of the invention or a compound of theinvention. Such a nucleic acid can be DNA or RNA; and is preferably DNAand can be in the form of a plasmid or vector. Reference is again forexample made to WO 08/020079 and to some of the other patentapplications of applicant/assignee cited herein.

The invention also relates to a composition that comprises at least oneVH domain of the invention, compound of the invention or nucleic acidencoding either of the same.

The invention further relates to a pharmaceutical composition thatcomprises an ISV (and preferably a therapeutic ISV) or a protein orpolypeptide comprising at least one ISV (and preferably at least onetherapeutic ISV), wherein said ISV, protein or polypeptide is as furtherdescribed herein (i.e. an ISV, protein or polypeptide according to oneor more of the aspects described herein, and in particular according toone or more of the aspects described on the preceding pages; and more inparticular an ISV, protein or polypeptide that has a C-terminalend/sequence that is according to one or more of the aspects describedherein), and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances. Such compositions, carriers, diluents or excipientscan for example be as described in WO 08/020079 for pharmaceuticalcompositions that comprise a Nanobody or a protein or polypeptide thatcomprises at least one Nanobody (and as already mentioned, according tothe present invention, the ISV is also preferably a Nanobody).

The invention further relates to an ISV or a protein or polypeptidecomprising at least one ISV for use in therapy of a disease in a humanbeing (e.g. a patient in need of such therapy), wherein said ISV,protein or polypeptide is as further described herein (i.e. an ISV,protein or polypeptide according to one or more of the aspects describedherein, and in particular according to one or more of the aspectsdescribed on the preceding pages; and more in particular an ISV, proteinor polypeptide that has a C-terminal end/sequence that is according toone or more of the aspects described herein).

The invention further relates to the use of an ISV or a protein orpolypeptide comprising at least one ISV in the preparation of apharmaceutical composition, wherein said ISV, protein or polypeptide isas further described herein (i.e. an ISV, protein or polypeptideaccording to one or more of the aspects described herein, and inparticular according to one or more of the aspects described on thepreceding pages; and more in particular an ISV, protein or polypeptidethat has a C-terminal end/sequence that is according to one or more ofthe aspects described herein).

The invention further relates to a method of treatment which comprisesadministering to a human subject (e.g to a patient in need of suchtreatment) an ISV or a protein or polypeptide comprising at least oneISV in the preparation of a pharmaceutical composition, wherein saidISV, protein or polypeptide is as further described herein (i.e. an ISV,protein or polypeptide according to one or more of the aspects describedherein, and in particular according to one or more of the aspectsdescribed on the preceding pages; and more in particular an ISV, proteinor polypeptide that has a C-terminal end/sequence that is according toone or more of the aspects described herein); or a pharmaceuticalcomposition (as described above) that comprises at least one such ISV,protein or polypeptide.

With respect to the above, it will be clear that the therapeutic use ofthe ISV's, proteins and polypeptides described herein are a veryimportant aspect of the invention, as such therapeutic use (or theclinical development of such ISV's, proteins and polypeptides for suchtherapeutic use) may involve the use of ADA assays to determine whethersaid ISV, protein or polypeptide is immunogenic (i.e. can give rise toADA's when administered to a human subject). In this respect, it willalso be clear that concerns about possible immunogenicity will inparticular have to be addressed when a therapeutic is either used forlonger periods of time (for during weeks, months or years), and/or has ahalf-life (preferably expressed as t½-beta) in a human subject of atleast 3 days, such as at least one week, and up to 10 days or more.

Thus, according to one specific aspect, the invention relates to an ISV,protein, polypeptide, compound or molecule of the invention as describedherein (or pharmaceutical composition the same) that is intended fortreatment of a chronic disease in a human being, and/or such ISV,protein, polypeptide as described herein is intended to be present inthe circulation of the subject (i.e. at pharmacologically active levels)to which it is administered (i.e. at a therapeutically active dose) forat least a period of one week, preferably at least two weeks, such as atleast a months; and/or such ISV, protein, polypeptide as describedherein is such that it has a half-life (preferably expressed as t½-beta)in a human subject of at least 3 days, such as at least one week, and upto 10 days or more; and/or such an ISV, protein, polypeptide orpharmaceutical composition as described herein is intended to beadministered to a human being as two or more doses that are administeredover a period of at least 3 days, such as at least one week, for exampleat least two weeks or at least one month, or even longer (i.e. at least3 months, at least 6 months or at least one year), or even chronicallyadministered.

Also, as will be clear to the skilled person based on the disclosureherein, the improvements to VH domains described herein and theresulting improved VH domains will find particular use in proteins,polypeptides or other compounds or molecules that are intended foradministration to human subjects (and in particular patients) whoseblood/serum contains (or is suspected to contain) pre-existingantibodies of the kind that—according to the present invention—have beenfound in samples obtained from SLE patients, i.e. pre-existingantibodies that can bind to the C-terminal region of a VH-domain even inthe presence of a C-terminal extension as described herein. Inparticular, the improvements to VH domains described herein and theresulting improved VH domains will find particular use in proteins,polypeptides or other compounds or molecules that are intended to treator prevent diseases or disorders in such patients. This may be anydisease or disorder, but may in particular be a disease or disorder thatresults in and/or is associated with the presence or emergence of suchpre-existing antibodies (one example being SLE, but it is expected thatother severe (auto-)-immune disorders may also lead to such pre-existingantibodies. This can be easily ascertained by testing samples obtainedfrom the relevant patient population for the presence of suchpre-existing antibodies, essentially in a manner analogous to the testsperformed on samples from SLE patients in the Experimental Part below).

Thus, according to one specific aspect, the invention relates to an ISV,protein, polypeptide, compound or molecule of the invention as describedherein (or a pharmaceutical composition the same) that is intended foradministration to a human subject whose blood contains pre-existingantibodies that can bind to the exposed C-terminal region of a VH domaineven when said VH-domain contains a C-terminal extension as describedherein (or where the C-terminal end of the VH domain is linked toanother protein or polypeptide, such as another ISV, optionally via asuitable linker).

In particular, the invention relates to an ISV, protein, polypeptide,compound or molecule of the invention as described herein (or apharmaceutical composition the same) for use in the treatment of adisease or disorder in a human subject whose blood contains pre-existingantibodies that can bind to the exposed C-terminal region of a VH domaineven when said VH-domain contains a C-terminal extension as describedherein (or where the C-terminal end of the VH domain is linked toanother protein or polypeptide, such as another ISV, optionally via asuitable linker). Said disease or disorder can be any disease ordisorder, but can in particular be a disease or disorder that leads to,results into or otherwise is associated with the presence of suchpre-existing antibodies in the blood of such a patient, such as SLE oranother (severe) autoimmune disease.

Thus, according to a more specific aspect, the invention relates to anISV, protein, polypeptide, compound or molecule of the invention asdescribed herein (or a pharmaceutical composition the same) for use inthe treatment of a disease or disorder in a human subject/patient,wherein said disease or disorder is a disease or disorder that leads to,results into or otherwise is associated with the presence ofpre-existing antibodies in the blood of said human subject/patient thatcan bind to the exposed C-terminal region of a VH domain even when saidVH-domain contains a C-terminal extension as described herein (or wherethe C-terminal end of the VH domain is linked to another protein orpolypeptide, such as another ISV, optionally via a suitable linker). Forexample, such an ISV, protein, polypeptide, compound or molecule of theinvention as described herein (or a pharmaceutical composition the same)for use in the treatment of SLE or another (severe) auto-immune diseasein a human subject/patient.

As will be clear to the skilled person, when a protein, polypeptide,compound or molecule is intended for the prevention or treatment of sucha disease or disorder, it will contain at least one (such as one, two,three or four) domains, binding units or moieties or entities that aretherapeutically active against the relevant disease or disorder (e.g.directed against a target or pathway that is therapeutically relevantfor the pertinent disease or disorder). Again, such binding domains orbinding units may for example be (other) ISVD's, and according to oneaspect may in particular be VH domains or ISVD's of the invention.Another general example of such a protein, polypeptide, compound ormolecule is a protein, polypeptide, compound or molecule in which saidone or more therapeutic domains, binding units or moieties or entitiesmay not be ISVD's (but for example derived from another scaffold), butthat contains a VH domain of the invention to extend the half-life ofthe same (such as a serum albumin binder as described herein).

In a further aspect, the VH domains, ISVD's or compounds of theinvention (as described herein) are directed towards the ion channelKv1.3. Some preferred, but non-limiting examples of such VH domains,ISVD's or compounds are given in Example 7, and the VH domains againstKv1.3 described in said example (as well as compounds of the inventioncomprising the same) as well as the specific compounds of the inventionagainst Kv1.3 described in said example form further aspects of thepresent invention.

For example and without limitation, a compound of the invention that isdirected towards Kv1.3 can comprise or essentially consist of a singleVH domain—and preferably Nanobody—of the invention directed againstKv1.3 or can comprise or essentially consist of at least two (such astwo or three) VH domains—and preferably Nanobodies—of the inventiondirected against Kv1.3. When such a polypeptide contains two or more VHdomains of the invention against Kv1.3, these VH domains can be the sameor different, and when they are different they can be directed againstthe same epitope on Kv1.3 or subunit on Kv1.3 or against differentepitopes or subunits.

Again, as generally described herein for the compounds of the invention,such compounds can suitably contain one or more linkers, can contain aC-terminal extension (i.e. as further described herein) and can alsocontain one or more further binding units or binding domains (or otheramino acid sequences or moieties), such as a further ISVD directedagainst a different target than Kv1.3. For example and withoutlimitation, the compounds of the invention can (also) contain a bindingdomain or binding unit that provides for extended half-life, such as anISVD against a serum protein such as serum albumin (for example, aNanobody against human serum albumin such as a Nanobody of the inventionagainst human serum albumin).

In further aspects, the invention relates to (synthetic) libraries ofimmunoglobulin variable domain sequences that are as described herein(i.e. containing the amino acid residues/mutations/substitutions asdescribed herein). Such libraries will usually contain at least 100different sequences, such as at least 1000 different sequences, inparticular more than 10⁵ different sequences, more in particular morethan 10⁶, such as 10⁸ to 10¹⁰ or more different sequences (meaning inits broadest sense, with at least one amino acid difference between thesequences), usually all with (essentially) the same framework sequences(said framework sequences containing the amino acid residues/mutationsindicated herein) and different CDRs (meaning that each sequence in thelibrary has at least “one amino acid difference” in at least one of itsCDRs compared to the other sequences in the library).

Synthetic libraries of immunoglobulin single variable domain sequences(for example based on human VH sequences or camelid-derived VHHsequences) and methods of generating/constructing them (includinglibraries based on pre-determined scaffolds and/or containing one ormore specific amino acid residues/mutations in the framework regions)are well known in the art. Reference is for example made to Tanha etal., J. Biol. Chem., Vol. 276, pp. 24774-24780, 2001; Bond et al., J.Mol. Biol. (2003) 332, 643-655; Mandrup et al., PLOS One, October 2013,Volume 8, Issue 10, e76834; Goldman et al., Anal. Chem., 2006, 78,8245-8255; Hussak et al., Protein Engineering, Design & Selection vol.25 no. 6 pp. 313-318, 2012; and Chen et al., Methods Mol. Biol., 2009,525, 81. The techniques described therein (and similar techniques knownper se) can be suitably used or adapted for generating theimmunoglobulin single variable domain libraries according to theinvention.

The ISVD's present in such libraries can suitably contain any suitableCDR's from any suitable source, such as CDR's obtained/generatedstarting from the immune repertoire from a “naïve” mammal (such as aspecies of camelid or human sequences), CDR's obtained/generatedstarting from the immune repertoire from an animal (such as a species ofcamelid) that has been suitably immunized with an antigen; a fullysynthetic CDR repertoire; or a repertoire that has been obtained throughtechniques such as mutagenesis (for example random mutagenesis orsite-specific mutagenesis). Such a library can for example also be alibrary that has been generated in the course of affinity maturationprocedures known per se.

The framework regions of the ISVD's present in such libraries can besuitably derived from any suitable starting sequence/scaffold, forexample based on a scaffold that has been derived starting from a VHsequence (such as a human VH sequence) or starting from a nanobodysequence (such as a VHH sequence or a humanized VH sequences). It isalso possible that a library contains ISVD's that are derived from twoor more different sources or that are based on two or more differentscaffolds (for example, because the library has been obtained bycombining two or more libraries obtained from different sources or basedon different scaffolds).

Also, (the ISVD's present in) the libraries may be in the form ofproteins or in the form of a DNA or RNA encoding the relevant ISVD's.For example, the libraries may be in the form of an expression librarysuitable for screening and/or selection techniques, and may for thispurpose for example be in a form that can be displayed using suitabledisplay techniques such as phage display library, a yeast displaylibrary or a ribosome display library.

Thus, generally, the invention also relates to libraries (as describedherein) containing VH domains of the invention (as further describedherein). Preferably, according to one specific aspect of such a library,the VH domains present all have the same (or essentially the same)framework sequences, but different CDR sequences (again, as mentioned,this means that each of the individual VH domains in the library has atleast one amino acid difference in at least one CDR compared to theother VH domains in the library).

In one aspect, such a library of the invention is a library of ISVD's ofthe invention (as further described herein, including a library ofsuitable nucleic acids encoding said ISVD's) in which:

the amino acid residue at position 11 is one of L, V or K; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of T, V or L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

in which either (i) the amino acid residue at position 112 is one of Kor Q; and/or (ii) the amino acid residue at position 89 is T; and/or(iii) the amino acid residue at position 89 is L and the amino acidresidue at position 110 is one of K or Q; and (iv) in each of cases (i)to (iii), the amino acid at position 11 is preferably V. Optionally, theISVD's present in such a library may also contain a C-terminal extension(as further described herein for the VH domains of the invention) and/orsuitably contain a suitable tag (such as a histidine tag).

In another aspect, such a library of the invention is a library ofISVD's of the invention (as further described herein, including alibrary of suitable nucleic acids encoding said ISVD's) in which:

the amino acid residue at position 11 is V; and

the amino acid residue at position 14 is one of A or P; and

the amino acid residue at position 41 is one of A or P; and

the amino acid residue at position 89 is one of L; and

the amino acid residue at position 108 is one of Q or L; and

the amino acid residue at position 110 is one of T, K or Q; and

the amino acid residue at position 112 is one of S, K or Q;

Optionally, the ISVD's present in such a library may also contain aC-terminal extension (as further described herein for the VH domains ofthe invention) and/or suitably contain a suitable tag (such as ahistidine tag).

The libraries of the invention may be used for any suitable/intendedpurpose known per se. For example, they may for example be used forscreening and/or selection purpose (or as part of a screening and/orselection process), be used for/as part of affinity maturation purposesor other processes intended to yield improved VH domains, or for examplefor alanine scanning. In practice, usually, the size, design and otherfeatures of the library will be adapted to its intended use, as will bewithin the skill of the artisan.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures, in which:

FIG. 1 is a table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT);

FIG. 2 lists the sequences referred to herein;

FIG. 3 is a plot showing data points obtained in Example 4 when 96 serumsamples were tested for binding a representative Nanobody with an S112Kmutation (Reference A+S 112K+C-terminal alanine, indicated as (2) inFIG. 3), compared to a reference Nanobody without an S112K mutation(Reference A, SEQ ID NO: 44, indicated as (1) in FIG. 3);

FIG. 4 is a plot showing data points obtained in Example 4 when 129serum samples were tested for binding a representative Nanobody with anV89T mutation (Reference A+L11V+V89T+C-terminal alanine, indicated as(2) in FIG. 4), compared to a reference Nanobody without an V89Tmutation (Reference A, SEQ ID NO: 44, indicated as (1) in FIG. 4);

FIG. 5 is a plot showing data points obtained in Example 5 when 100serum samples were tested for binding representative Nanobodies withV89L, T110K and/or T110Q mutations (Reference A+L11V+V89L+C-terminalAla, indicated as (2) in FIG. 5; Reference A+L11V+V89L+T110K+C-terminalAla, indicated as (3) in FIG. 5; Reference A+L11V+V89L+T110Q+C-terminalAla, indicated as (4) in FIG. 5) and ReferenceA+L11V+T87A+V89L+C-terminal Ala, indicated as (5) in FIG. 5), comparedto a reference Nanobody without any of these mutations (Reference A, SEQID NO: 44, indicated as (1) in FIG. 5).

FIG. 6 is a plot showing data points obtained in Example 6 when 98 serumsamples obtained from healthy volunteers were tested for bindingrepresentative trivalent Nanobody constructs. Each dot represents a datapoint collected by testing of the indicated construct against one of the98 serum samples. Legenda: (1)=Reference X (Nanobody A-35GS-NanobodyA-35GS-Nanobody B); (2)=Reference X+C-terminal Ala; (3)=ReferenceX+L11V+V89L+C-terminal Ala; (4)=Reference X+L11V+T87A+V89L+C-terminalAla; (5)=Reference X+L11V+V89L+T110K+C-terminal Ala; (6)=ReferenceX+L11V+V89L+T110Q+C-terminal Ala.

FIG. 7 is a plot showing data points obtained in Example 6 when 30 serumsamples obtained from healthy volunteers (samples selected for hightiters of pre-existing antibodies or presence of pre-existing antibodieswith high degree of binding even in the presence of a C-terminal alanineextension) were tested for binding representative trivalent Nanobodyconstructs. Each dot represents a data point collected by testing of theindicated construct against one of the 30 serum samples. Legenda:(1)=Reference X+C-terminal Ala; (2)=Reference X+L11V+V89L+C-terminalAla; (4)=Reference X+L11V+T87A+V89L+C-terminal Ala; (4)=ReferenceX+L11V+V89L+T110K+C-terminal Ala.

FIG. 8 is a plot showing data points obtained in Example 6 when 98 serumsamples obtained from healthy volunteers were tested for bindingrepresentative bivalent Nanobody constructs. Each dot represents a datapoint collected by testing of the indicated construct against one of the30 serum samples. Legenda: (1)=Reference Y (Nanobody A-35GS-Nanobody B);(2)=Reference Y+C-terminal Ala; (3)=Reference Y+L11V+V89L+C-terminalAla; (4)=Reference Y+L11V+T87A+V89L+C-terminal Ala; (5)=ReferenceY+L11V+V89L+T110K+C-terminal Ala; (6)=ReferenceY+L11V+V89L+T110Q+C-terminal Ala;

FIGS. 9A and 9B show preferred but non-limiting examples of monovalentNanobodies of the invention (FIG. 9A) and trivalent bispecific half-lifeextended compounds of the invention (FIG. 9B) against the ion channelKv1.3; and FIG. 9C lists some preferred CDRs (classification accordingto Kabat and Abm, respectively) for ISVDs against Kv1.3.

FIG. 10 is a plot showing data points obtained in Example 7 when 47serum samples obtained from diabetic human subjects were tested forbinding representative trivalent bispecific half-life extended compoundsof the invention against Kv1.3. Each dot represents a data pointcollected by testing of the indicated construct against one of the 47serum samples. The SEQ ID NO's refer to the relevant sequences listed inFIG. 9;

FIG. 11 is a plot showing data points obtained in Example 7 when 90serum samples obtained from healthy volunteers were tested for bindingrepresentative trivalent bispecific half-life extended compounds of theinvention against Kv1.3. Each dot represents a data point collected bytesting of the indicated construct against one of the 47 serum samples.The SEQ ID NO's refer to the relevant sequences listed in FIG. 9.

FIGS. 12A and 12B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against IL-23that are based on the indicated reference sequences. See also Example 8;

FIG. 13 lists the CDR's and amino sequences of some preferred, butnon-limiting examples of Nanobodies of the invention against OX40-L thatare based on the indicated reference sequence. See also Example 9;

FIG. 14 lists the CDR's and amino sequences of some preferred, butnon-limiting examples of Nanobodies of the invention against IgE thatare based on the indicated reference sequence. See also Example 10;

FIGS. 15A and 15B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against CXCR-4that are based on the indicated reference sequences. See also Example11;

FIGS. 16A and 16B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against HER-3that are based on the indicated reference sequences. See also Example12;

FIGS. 17A and 17B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against TNFthat are based on the indicated reference sequences. See also Examples13 and 14;

FIGS. 18A and 18B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against c-Metthat are based on the indicated reference sequences. See also Example15;

FIG. 19 lists the CDR's and amino sequences of some preferred, butnon-limiting examples of Nanobodies of the invention against RANK-L thatare based on the indicated reference sequence. See also Example 16;

FIGS. 20A to 20C list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against CXCR-7that are based on the indicated reference sequences. See also Example17;

FIGS. 21A and 21B list the CDR's and amino sequences of some preferred,but non-limiting examples of Nanobodies of the invention against A-betathat are based on the indicated reference sequences. See also Example18;

FIG. 22 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against IL-23;

FIG. 23 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against OX40-L;

FIG. 24 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against IgE;

FIG. 25 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against CXCR-4;

FIG. 26 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against HER-3;

FIG. 27 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against TNF;

FIGS. 28A and 28B give the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against c-Met;

FIG. 29 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against RANK-L;

FIG. 30 gives the amino acid sequences of some preferred butnon-limiting examples of compounds of the invention against A-beta;

FIG. 31A is a plot showing data points obtained in Example 19 when 92serum samples obtained from healthy volunteers were tested for bindingrepresentative trivalent bispecific half-life extended compounds of theinvention against A-beta. Each dot represents a data point collected bytesting of the indicated construct against one of the 92 serum samples.The reference numbers are as listed in Table CC-1. Similarly, FIG. 31Bis a plot showing data points obtained in Example 19 when 92 serumsamples obtained from healthy volunteers were tested for binding the(monovalent) C-terminal Nanobodies that are present in the constructstested in FIG. 31A. Each dot represents a data point collected bytesting of the indicated construct against one of the 92 serum samples.The reference numbers are as listed in Table CC-2.

EXPERIMENTAL PART

The human samples used in the Experimental Part below were eitherobtained from commercial sources or from human volunteers (after allrequired consents and approvals were obtained) and were used inaccording with the applicable legal and regulatory requirements(including but not limited to those regarding medical secret and patientprivacy)

In the Examples below, the binding of pre-existing antibodies that arepresent in the samples used (i.e. from healthy volunteers, rheumatoidarthritis (RA) patients and SLE patients) to the Nanobodies tested wasdetermined using ProteOn as follows: Binding of pre-existing antibodieson Nanobodies captured on human serum albumin (HSA) was evaluated usingthe ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS/Tween (phosphatebuffered saline, pH7.4, 0.005% Tween20) was used as running buffer andthe experiments were performed at 25° C. The ligand lanes of a ProteOnGLC Sensor Chip were activated with EDC/NHS (flow rate 30 μl/min) andHSA was injected at 10 μg/ml in ProteOn Acetate buffer pH4.5 (flow rate100 μl/min) to render immobilization levels of approximately 3200 RU.After immobilization, surfaces were deactivated with ethanolamine HCl(flow rate 30 μl/min). Nanobodies were injected for 2 minutes at 45μl/min over the HSA surface to render a Nanobody capture level ofapproximately 200 RU. The samples containing pre-existing antibodieswere centrifuged for 2 minutes at 14,000 rpm and supernatant was diluted1:10 in PBS-Tween20 (0.005%) before being injected for 2 minutes at 45μl/min followed by a subsequent 400 seconds dissociation step. Aftereach cycle (i.e. before a new Nanobody capture and blood sampleinjection step) the HSA surfaces were regenerated with a 2 minuteinjection of HCl (100 mM) at 45 μl/min. Sensorgram processing and dataanalysis was performed with ProteOn Manager 3.1.0 (Bio-Rad Laboratories,Inc.). Sensorgrams showing pre-existing antibody binding were obtainedafter double referencing by subtracting 1) Nanobody-HSA dissociation and2) non-specific binding to reference ligand lane. Binding levels ofpre-existing antibodies were determined by setting report points at 125seconds (5 seconds after end of association). Percentage reduction inpre-existing antibody binding was calculated relative to the bindinglevels at 125 seconds of a reference Nanobody.

Example 1: S112K Mutation Inhibits Binding of Pre-Existing Antibodies

The influence of a substitution at position 112 on the binding ofpre-existing antibodies in human samples to Nanobodies was determinedand compared to the influence of a C-terminal alanine extension asdescribed in WO 12/175741.

Two reference compounds (Reference A without a C-terminal alanineextension and Reference B with a C-terminal alanine extension) andvariants of these reference compounds with different mutations atposition 112 were tested against sera obtained from six different RApatients and eight sera obtained from different healthy human subjects.Binding of pre-existing antibodies in the samples to the Nanobodiestested was measured on ProteOn according to the general protocoloutlined above. The results are shown in Table A below.

As can be seen, of the mutations at position 112 that were tested, theS112K mutation gave a reduction of binding of the pre-existingantibodies that were present in the tested sera that was comparable tothat of the C-terminal alanine extension (even without the C-terminalalanine extension being present in the S112K variant). Similar resultswere obtained with three human plasma samples (data not shown).

TABLE A comparison of mutations at position 112 to a C-terminal alanineextension on binding of pre-existing antibodies present in sera from RApatients and human volunteers RA sera healthy human sera Average bindingon Reference A (RU) Reference A 75 96 44 11 117 141 242 81 121 179 93 9291 92 Inhibition compared to average binding on Reference A (%)Reference A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Reference B 70 90 100 88 84 9753 78 83 93 93 81 86 86 Reference A (S112E) 51 67 63 65 66 84 31 82 7259 73 100 74 70 Reference A (S112F) 75 51 85 100 79 39 56 86 69 60 78 9376 79 Reference A (S112K) 88 88 94 100 94 100 86 93 87 87 95 100 84 91Reference A (S112L) 69 45 63 54 50 81 37 74 59 42 75 97 68 83

Example 2: Influence of S112K Mutation on Binding of Pre-ExistingAntibodies that are Present in Human SLE Samples

The same Nanobody variants as used in Example 1 were tested for bindingby pre-existing antibodies from 7 serum samples obtained from patientswho were confirmed positive for systemic lupus erythemathosus (SLE). Forcomparison, plasma samples from two healthy human volunteers wereincluded.

Binding of pre-existing antibodies in the samples to the Nanobodiestested was measured on ProteOn according to the general protocoloutlined above. The results are shown in Table B below.

As can be seen from a comparison of the binding data for Reference A andReference B and nanobodies of the invention, the samples obtained fromsome of the SLE patients appear to contain certain pre-existingantibodies that can still bind to Nanobodies even in the presence of aC-terminal alanine residue (the C-terminal alanine residue didessentially prevent/remove (partially or essentially fully) all bindingof the pre-existing antibodies that were present in the plasma samplesfrom healthy volunteers).

It can further be seen that the binding of these pre-existing antibodiesfrom SLE samples could be greatly reduced by mutations at positions 11and 112 (and in case of position 112, in particular S112K).

TABLE B comparison of mutations at positions 11 and 112 to a C-terminalalanine extension on binding of pre-existing antibodies present in serafrom SLE patients Plasma samples obtained from healthy Serum samplesobtained from SLE patients volunteers Average binding on Reference A(RU) Reference A 45 61 38 40 43 20 69 128 171 Inhibition compared tobinding on Reference A (%) Reference B 20 16 13 45 53 86 101 95 90Reference A (L11E) 63 88 117 61 87 88 92 68 21 Reference A (L11K) 87 97107 54 106 79 102 100 61 Reference A (L11V) 68 84 49 56 95 91 21 23 6Reference A (L11Y) 27 71 111 37 84 74 72 13 3 Reference A (S112E) 13 5691 77 74 91 94 84 22 Reference A (S112F) −6 18 26 −13 62 69 117 74 43Reference A (S112K) 71 77 105 80 116 86 120 87 62 Reference A (S112L)−36 36 48 −24 123 19 84 91 3

Example 3: Influence of Combined Framework Mutations and C-TerminalExtensions on Binding of Pre-Existing Antibodies that are Present inHuman SLE Samples

Four different Nanobodies (with specific framework mutations and with orwithout C-terminal alanine extension) were tested for binding ofpre-existing antibodies from 5 serum samples obtained from patients whowere confirmed positive for systemic lupus erythemathosus (SLE). Forcomparison, one plasma sample from a healthy human volunteer wasincluded.

Binding of pre-existing antibodies in the samples to the Nanobodiestested was measured on ProteOn according to the general protocoloutlined above. The results are shown in Tables C and D below.

As can be seen from a comparison of the binding data for Reference A andReference B, the samples obtained from SLE patients appear to contain acertain pre-existing antibodies that can still bind to Nanobodies evenin the presence of a C-terminal alanine residue (the C-terminal alanineresidue did essentially prevent/remove all binding of the pre-existingantibodies that were present in the plasma samples from the healthyvolunteer).

It can further be seen that the binding of these pre-existing antibodiesfrom SLE samples could be greatly reduced by mutations at positions 11and 112 (and in case of position 112, in particular S112K).

Example 4: Influence of a V89T Mutation on Binding of Pre-ExistingAntibodies in Samples from SLE Patients

As described herein, samples obtained from certain SLE patients appearto contain pre-existing antibodies/factors that can bind to the exposedC-terminal end of a VH domain, even when a C-terminal extension ispresent. It was investigated whether a V89T mutation could reduce orprevent/remove such binding, with or without the presence of aC-terminal extension. The results are also shown in Tables C and Ebelow.

As can be seen, a V89T mutation could essentially prevent/remove bindingof pre-existing antibodies that are present in samples obtained from SLEpatients, to a similar degree as an S112K mutation. However, as can beseen from comparing the data given in Tables C and E for nanobodies witha V89T mutation without a C-terminal extension with similar nanobodieswith an S112K mutation and without a C-terminal extension, having amutation at position 112 in a nanobody without a C-terminal extensiongenerally reduces binding of pre-existing antibodies in samples from ahealthy volunteer to a larger degree than a V89T mutation (i.e. 100%,85% and 64% of S112K nanobodies vs. 9%, 11% and 16% for V89T nanobodies,respectively). For this reason, the use of a mutation at position 112(and in particular S112K or S112K) will often be preferred over the useof a mutation at position 89 (such as V89T).

However, as can also be seen from the data in Tables C and E, adding aC-terminal alanine to a V89T nanobody completely prevented/removed thebinding of pre-existing antibodies in a sample obtained from healthyvolunteers, and for this reason a combination of a V89T mutation and aC-terminal extension as described herein will usually be preferred (i.e.over the use of a V89T without C-terminal extension) if the V89Tnanobody or VH domain has, or is intended to have, an exposed C-terminalregion in the protein or polypeptide in which it will be present (forexample, because if forms the C-terminal end of the same).

TABLE C influence of different mutations of binding by pre-existingantibodies in samples obtained from SLE patients and human volunteersSample obtained from healthy Mutation(s) Samples obtained from SLEpatients volunteer L11K L11V V89T S112K 114A SLE25 SLE37 SLE39 SLE41NB13025-14 004-030-ABL-02 Average binding to Reference A Average bindingfor 38 66 30 41 45 175 Reference A Inhibition compared to averagebinding to Reference A captured on HSA (%) Reference A + V89T, no x x100 98 100 100 98 9 C-terminal extension Reference A + V89T + x x x 9798 100 98 100 100 C-terminal alanine Reference A + S112K, X x 100 100100 100 98 100 no C-terminal extension Reference A + S 112K + X x x 100100 100 99 99 100 C-terminal alanine (*) (*) Note: this Nanobody wasalso used to generate the data shown in FIG. 3 and Table F (see below)

TABLE D influence of different mutations of binding by pre-existingantibodies in samples obtained from SLE patients and human volunteersSample obtained from healthy Mutation Samples obtained from SLE patientsvolunteer L11V V89L S112Q 114A SLE25 SLE37 SLE39 SLE41 NB13035-14004-030-ABL-02 Average binding to Reference A Average binding forReference A ND 71 51 ND 41 180 Inhibition compared to average binding toReference A captured on HSA (%) Reference A + V89L + S 112Q + x x x ND100 100 ND 100 97 C-terminal alanine Reference A + L11V + S 112Q + x x xND 100 100 ND 100 99 C-terminal alanine Reference A + S 112Q +C-terminal x x ND 92 85 ND 94 100 alanine

TABLE E influence of different mutations on binding by pre-existingantibodies in samples from SLE patients and human volunteers Sample fromMutation(s) Samples obtained from SLE patients healthy volunteer L11VV89L V89T S112K 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02Average binding to Reference A Reference A 28 44 26 33 30 151 Inhibitioncompared to average binding to Reference A captured on HSA (%) ReferenceA + V89L, no C-terminal x 77 64 53 63 41 35 extension Reference A +V89L + C-terminal x x 35 27 63 42 46 83 alanine Reference A + V89T, noC-terminal x 68 12 84 100 71 11 extension Reference A + V89T +C-terminal x x 46 35 71 100 97 99 alanine Reference A + V89T + L11V, nox x 100 97 100 100 100 16 C-terminal extension Reference A + V89T +L11V + x x x 100 100 100 100 100 67 C-terminal alanine (*) Reference A +S112K + V89L, no x x 100 100 100 100 100 85 C-terminal extensionReference A + S112K + V89L x x x 100 100 100 100 100 100 C-terminalalanine Reference A + S112K + L11V, no x x 100 100 100 100 100 64C-terminal extension Reference A + S112K + L11V + x x x 100 100 100 100100 100 C-terminal alanine (*) Note: this Nanobody was also used togenerate the data shown in FIG. 4 and Table G (see below)

To confirm that the results/findings from the table above are broadlyapplicable, representative Nanobodies with S112K and/or V89T mutationswere tested against a test panel of 96 (S112k) and 129 (V89T) humanserum samples. Binding was determined on ProteOn using the protocol setout above.

The results are summarized FIG. 3 and Table F (representative Nanobodywith an S112K mutation) and FIG. 4 and Table G (representative Nanobodywith a V89T mutation).

In FIG. 3, a Nanobody with an S112K mutation (ReferenceA+S112K+C-terminal alanine—see Table C above) was compared to areference Nanobody (Reference A; SEQ ID NO:44). The Nanobody with theS112K mutation and Reference A were both tested against each of theserum samples, and the binding level at 125 seconds (RU) was determined.The data was then plotted in FIG. 3, with each point presenting thebinding measured in one sample for either Reference A (indicated as (1)in FIG. 3) or for the S112K mutant (indicated as (2) in FIG. 3). Thedotted line indicates a measured binding level of 20 RU.

The same data is also represented numerically in Table F, whichmentions—for Reference A and the S112K mutant, respectively—the totalnumber of samples tested that gave a level of binding at 125 seconds ofmore than 20 RU, less than 20 RU (i.e. between 0 and 20 RU) and lessthan 10 RU.

As can be seen from the data plotted in FIG. 3 and shown in Table F, forReference A, more than half of the 96 samples tested gave a bindinglevel of more than 20 RU (in some cases, as high as 150-200 RU),indicating that the pre-existing antibodies present in the sample werebinding to Reference A. By comparison, for the S112K mutant, no samplegave a binding level of more than 20 RU (and most less than 10 RU),indicating that the S122K mutation was essentially capable ofreducing/preventing binding of the pre-existing antibodies in all of the96 samples tested.

A similar plot and similar data is shown in FIG. 4 and Table G,respectively, for a representative Nanobody with a V89T mutation(Reference A+L11V+V89T+C-terminal alanine; see Table E above), testedagainst 129 serum samples and again compared to Reference A (indicatedby (1) in FIG. 4; the V89T mutant is indicated by (2) in FIG. 4). Again,from the plot in FIG. 4 and the data in Table G, it can be seen thatwith a few exceptions (i.e. less than 10% of samples tested, which theneach gave an absolute binding value after 125 seconds of about 100 RU orless), the V89T mutation was capable of reducing/preventing binding ofthe pre-existing antibodies in most of the 129 samples tested, whereasthe reference without the V89T mutation was bound by pre-existingantibodies in most of the samples tested.

TABLE F testing of representative Nanobody with S112K mutation against96 serum samples. Binding level Binding level Binding level Nanobody at125 at 125 at 125 tested seconds < 10 RU seconds < 20 RU seconds > 20 RUReference A 34 41 55 Reference A + 92 96 0 S112K + C-terminal alanine

TABLE G testing of Nanobody mutants with V89L and/or T100K/Q mutationsagainst 129 serum samples. Binding level at 125 Binding level at 125Binding level at 125 Nanobody tested seconds < 10 RU seconds < 20 RUseconds > 20 RU Reference A 27 40 89 Reference A + L11V + 110 119 10V89T + C- terminal alanine

Example 5: Influence of a V89L Mutation Combined with a T110K or T110QMutation on Binding of Pre-Existing Antibodies in Samples from SLEPatients

As described herein, samples obtained from certain SLE patients appearto contain pre-existing antibodies/factors that can bind to the exposedC-terminal end of a VH domain, even when a C-terminal extension ispresent. It was investigated whether a V89L and/or a T110Q or T110Kmutation (or a combination thereof) could reduce or prevent/remove suchbinding, with or without the presence of a C-terminal extension. Theresults are also shown in Tables H and I, which present the data fromtwo separate experiments (Table D above also shows data for an S112Qmutation of the invention combined with a V89L mutation). The Nanobodiesused in Table I were also used in generating the data shown in FIG. 5and Table J.

TABLE H influence of different mutations on binding by pre-existingantibodies in samples from SLE patients and human volunteers Sample fromMutation(s) Samples obtained from SLE patients healthy volunteer L11VV89L T110K T110Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02Average binding to Reference A Reference A 28 44 26 33 30 151 Inhibitioncompared to average binding to Reference A captured on HSA (%) ReferenceA + V89L, no C-terminal x 77 64 53 63 41 35 extension Reference A +V89L + C-terminal x x 35 27 63 42 46 83 alanine Reference A + L11V +V89L, no x x 100 100 8 95 100 29 C-terminal extension Reference A +L11V + V89L + x x x 94 70 100 78 100 89 C-terminal alanine (*) ReferenceA + V89L + T110K, no C- x x 100 77 100 100 100 65 terminal extensionReference A + V89L + T110K, x x x 96 70 100 100 100 100 C-terminalalanine Reference A + L11V + T110K, no C- x x 100 0 100 100 100 43terminal extension Reference A + L11V + T110K + x x x 96 0 100 100 10099 C-terminal alanine (*) This Nanobody was also used in generating thedata shown in FIG. 5 and Table J

TABLE I influence of different mutations on binding by pre-existingantibodies in samples from SLE patients and human volunteers.Mutation(s) Samples obtained from SLE patients L11V T87A V89L T110KT110Q 114A SLE25 SLE37 SLE39 SLE41 NB13025-14 Average binding toReference A Reference A 50 72 55 56 58 Inhibition compared to averagebinding to Reference A captured on HSA (%) Reference A + L11V + V89L + xx x 100 100 98 100 99 C-terminal alanine Reference A + L11V + V89L +T110K + C- x x x x 100 81 98 99 98 terminal alanine Reference A + L11V +V89L + T110Q + C- x x x x 100 100 87 98 81 terminal alanine ReferenceA + L11V + T87A + V89L + C- x x x x 100 100 100 100 100 terminal alanine

To confirm that the results/findings from the table above are broadlyapplicable, representative Nanobodies with V89L, T110K and/or T110Qmutations were again tested against a panel of 99 human serum samples.Binding data was again obtained and plotted as indicated in Example 4for the results and data shown in FIGS. 3 and 4 and Tables F and G.

The Nanobodies tested were (the numbers correspond to the numbering usedin FIG. 5): (1) Reference A; (2) Reference A+L11V+V89L+C-terminal Ala;(3) Reference A+L11V+V89L+T110K+C-terminal Ala; (4) ReferenceA+L11V+V89L+T110Q+C-terminal Ala; (5) ReferenceA+L11V+T87A+V89L+C-terminal Ala. The results are shown in FIG. 5 andTable I.

As can be seen, introducing the mutations tested again greatly reducedthe number of samples in which the pre-existing antibodies were capableof binding the Nanobody tested. It can also be seen that, for Nanobody(2) in FIG. 5 (Reference A+L11V+V89L+C-terminal Ala), still some of thesamples showed binding of pre-existing antibodies after 125 seconds atlevels of more than 20 RU (but already much less than 100 RU). However,when the V89L mutation was combined with a T110K mutation (Nanobody (3))or a T100Q mutation (Nanobody (4)), then essentially all of the 99samples tested showed a binding level of less than 20 RU (and in factless than 10 RU, see Table J).

TABLE J testing of representative Nanobodies with V89L, T110K and/orT110Q mutations against 99 serum samples. Binding level at 125 Bindinglevel at 125 Binding level at 125 Nanobody tested seconds <10 RU seconds<20 RU seconds >20 RU Reference A 52 64 35 Reference A + L11V + 94 95 4V89L + C- terminal Ala Reference A + L11V + 99 99 0 V89L + T110K +C-terminal Ala Reference A + L11V + 99 99 0 V89L + T110Q + C-terminalAla Reference A + L11V + 99 99 0 T87A + V89L + C-terminal Ala

Example 6: Testing of Multivalent Constructs for Binding of Pre-ExistingAntibodies

Multivalent constructs are made based on the following nanobodies:

Nanobody A (directed against a therapeutic target): (SEQ ID NO: 92)EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSSNanobody B (directed against serum albumin): (SEQ ID NO: 93)EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSSNanobody C (directed against a therapeutic target): (SEQ ID NO: 94)EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFN KYVTSRDTWGQGTLVTVSS

The constructs made are listed in Table K below (with in each case, theintroduced substitution(s) in the relevant nanobody (if any) mentionedbetween parentheses. “HIS6” denotes an N-terminal his-tag of 6 histidineresidues, and “-Ala” denotes a C-terminal extension of 1 alanineresidue).

TABLE K multivalent constructs tested HIS6-Nanobody A(E1D, L11V,S112K)-35GS-Nanobody B-Ala HIS6-Nanobody A(E1D, L11V,S112K)-35GS-Nanobody B(L11V, S112K)-Ala HIS6-NanobodyA(E1D)-35GS-Nanobody B HIS6-Nanobody A(E1D)-35GS-Nanobody B-AlaHIS6-Nanobody A(E1D, L11V, S112K)-35GS-Nanobody B(L11V, S112K)HIS6-Nanobody A(E1D, S112K)-35GS-Nanobody B(S112K)-Ala HIS6-NanobodyA(E1D, S112Q)-35GS-Nanobody B(S112Q)-Ala HIS6-Nanobody A(E1D, V89L,S112K)-35GS-Nanobody B(V89L, S112K)-Ala HIS6-Nanobody A(E1D, L11V,S112Q)-35GS-Nanobody B(L11V, S112Q)-Ala HIS6-NanobodyB(E1D)-35GS-Nanobody A HIS6-Nanobody B(E1D)-35GS-Nanobody A-AlaHIS6-Nanobody A(E1D)-35GS-Nanobody B(L11V, S112K)-Ala HIS6-NanobodyB(E1D, L11V, S112K)-35GS-Nanobody A-Ala HIS6-NanobodyB(E1D,)-35GS-Nanobody A(L11V, S112K)-Ala HIS6-Nanobody A(E1D, V89L,S112Q)-35GS-Nanobody B(V89L, S112Q)-Ala HIS6-Nanobody B(E1D, L11V,S112K)-35GS-Nanobody A(L11V, S112K)-Ala HIS6-Nanobody B(S112K)-AlaNanobody B(L11V, S112K)-Ala Nanobody B(L11V, S112Q)-Ala NanobodyB(S112Q)-Ala HIS6-Nanobody A(E1D, S112K)-35GS-NanobodyA(S112K)-35GS-Nanobody B(S112K)- Ala HI56-Nanobody A(E1D,S112Q)-35GS-Nanobody A(S112Q)-35GS-Nanobody B(S112Q)- Ala HIS6-NanobodyA(E1D,L11V,S112K)-35GS-Nanobody A(L11V, S112K)-35GS-Nanobody B(L11V,S112K)-Ala HIS6-Nanobody A(E1D,V89L, S112K)-35GS-Nanobody A(V89L,S112K)-35GS- Nanobody B(V89L, S112K)-Ala HIS6-NanobodyA(E1D)-35GS-Nanobody A-35GS-Nanobody B HIS6-NanobodyA(E1D)-35GS-Nanobody A-35GS-Nanobody B-Ala NanobodyC(E1D)-9GS-AlaLB11-9GS-Nanobody C HIS6-Nanobody A(E1D, L11V,S112K)-35GS-Nanobody A(L11V, S112K)-35GS- Nanobody B-Ala HIS6-NanobodyC(E1D, L11V, S112K)-9GS-Nanobody B(L11V, S112K)-9GS-Nanobody C(L11V,S112K)-Ala Nanobody C(E1D)-9GS-AlaLB11-9GS-Nanobody C-Ala HIS6-NanobodyA(E1D, L11V, S112Q)-35GS-Nanobody A(L11V, S112Q)-35GS- Nanobody B(L11V,S112Q)-Ala HIS6-Nanobody A(E1D, V89L, S112Q)-35GS-Nanobody A(V89L,S112Q)-35GS- Nanobody B(V89L, S112Q)-Ala HIS6-Nanobody C(E1D,S112K)-9GS-Nanobody B(S112K)-9GS-Nanobody C(S112K)-Ala HIS6-NanobodyC(E1D, S112Q)-9GS-Nanobody B(S112Q)-9GS-Nanobody C(S112Q)-AlaHIS6-Nanobody C(E1D, L11V, S112Q)-9GS-Nanobody B(L11V,S112Q)-9GS-Nanobody C(L11V, S112Q)-Ala HIS6-Nanobody C(E1D, V89L,S112K)-9GS-Nanobody B(V89L, S112K)-9GS-Nanobody C(V89L, S112K)-AlaHIS6-Nanobody C(E1D, V89L, S112Q)-9GS-Nanobody B(V89L,S112Q)-9GS-Nanobody C(V89L, S112Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P,R39Q, K83R, V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L,K83R, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R,T87A, V89L, T91Y, Q108L)-35GS- Nanobody B (L11V, R30S, E44G, P45L, K83R,T87A, V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R,V89L, T91Y, Q108L, T110Q)-35GS- Nanobody B (L11V, R30S, E44G, P45L,K83R, V89L, Q108L, T110Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q,K83R, V89L, T91Y, Q108L, T110K)-35GS- Nanobody B (L11V, R30S, E44G,P45L, K83R, V89L, Q108L, T110K)-Ala HIS6-Nanobody A(E1D, L11V, A14P,R39Q, K83R, V89L, T91Y, Q108L)-35GS-Nanobody A(E1D, L11V, A14P, R39Q,K83R, V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R,V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, T87A,V89L, T91Y, Q108L)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, T87A,V89L, T91Y, Q108L)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, T87A,V89L, Q108L)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L,T91Y, Q108L, T110Q)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L,T91Y, Q108L, T110Q)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L,Q108L, T110Q)-Ala HIS6-Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L,T91Y, Q108L, T110K)-35GS- Nanobody A(E1D, L11V, A14P, R39Q, K83R, V89L,T91Y, Q108L, T110K)-35GS-Nanobody B(L11V, R30S, E44G, P45L, K83R, V89L,Q108L, T110K)-Ala

Representative multivalent constructs were tested for binding bypre-existing antibodies that are present in a blood or serum sampleobtained from patients with SLE and healthy volunteers. Both weredetermined using ProteOn, essentially as described above.

The representative constructs tested are listed in Tables L (trivalentconstructs) and M (bivalent constructs), and the results are shown inFIGS. 6-8 and Tables N-Q. The trivalent constructs tested were derivedfrom the reference construct Nanobody A-35GS-Nanobody A-35GS-Nanobody B(“Reference X”) and the bivalent constructs were derived from thereference construct Nanobody A-35GS-Nanobody B (“Reference X”). All ofthe constructs (except for the reference constructs) had, wereindicated, a C-terminal alanine residue as well as, in each of the“Nanobody A” and “Nanobody B” building blocks, the indicated mutations.

TABLE L trivalent constructs tested. Trivalent constructs The constructs(except for the reference constructs) had, were indicated, a C-terminalalanine residue as well as, in each of the building blocks, theindicated mutations. Mutation(s) L11V T87A V89L T110K T110Q 114AReference X (=Nanobody A-35GS-Nanobody A-35GS-Nanobody B) Reference X +x C-terminal Ala Reference X + L11V + x x x V89L C-terminal AlaReference X + L11V + x x x x V89L + T110K + C-terminal Ala Reference X +L11V + x x x x T87A + V89L + C-terminal Ala Reference X + L11V + x x x xV89L + T110Q + C-terminal Ala

TABLE M bivalent constructs tested. Bivalent constructs The constructs(except for the reference constructs) had, were indicated, a C-terminalalanine residue as well as, in each of the building blocks, theindicated mutations. Mutation(s) L11V T87A V89L T110K T110Q 114AReference Y (=Nanobody A-35GS-Nanobody B) Reference Y + x C-terminal AlaReference Y + L11V + x x x V89L + C-terminal Ala Reference Y + L11V + xx x x V89L + T110K + C-terminal Ala Reference Y + L11V + x x x x T87A +V89L + C-terminal Ala Reference Y + L11V + x x x x V89L + T110Q +C-terminal Ala

TABLE N results of testing trivalent constructs for binding bypre-existing antibodies present in 98 serum samples obtained fromhealthy human volunteers. Results are also represented in FIG. 6 as aplot in which each dot represents a data point collected by testing ofthe indicated construct against one of the 98 serum samples. Lane numberBinding level Binding level Binding Nanobody tested in the plot at 125at 125 level at 125 on 98 samples shown in seconds < 10 seconds < 20seconds > 20 (healthy subjects) FIG. 6 RU RU RU Reference X (1) 38 48 50Reference X + (2) 64 75 23 C-terminal Ala Reference X + (3) 94 95 3L11V + V89L + C-terminal Ala Reference X + (5) 95 96 2 L11V + V89L +T110K + C-terminal Ala Reference X + (6) 95 96 2 L11V + V89L + T110Q +C-terminal Ala Reference X + (4) 92 95 3 L11V + T87A +V89L + C-terminalAla

TABLE O results of testing trivalent constructs for binding bypre-existing antibodies present in 30 selected serum samples obtainedfrom healthy human volunteers. The 30 samples used were pre-selected foreither having a known high titer of pre-existing antibodies or becauseit was known that the pre-existing antibodies present in the samplestill has a high degree of binding even if a C-terminal alanine residueis present. Results are also represented in FIG. 7 as a plot in whicheach dot represents a data point collected by testing of the indicatedconstruct against one of the selected 30 serum samples. Nanobody Lanenumber Binding level Binding level Binding level tested on in the plotat 125 at 125 at 125 selected set shown in seconds < 10 seconds < 20seconds > 20 of 30 samples FIG. 7 RU RU RU Reference X + (1) 12 13 17C-terminal Ala Reference X + (2) 18 22 8 L11V + V89L + C-terminal AlaReference X + (3) 27 27 3 L11V + T87A + V89L + C-terminal Ala ReferenceX + (4) 26 26 4 L11V + V89L + T110K + C- terminal Ala

TABLE P results of testing bivalent constructs for binding bypre-existing antibodies present in 98 serum samples obtained fromhealthy human volunteers. Results are also represented in FIG. 8 as aplot in which each dot represents a data point collected by testing ofthe indicated construct against one of the 98 serum samples. Nanobodytested on 98 Lane number Binding level Binding level Binding levelsamples in the plot at 125 at 125 at 125 (healthy shown in seconds < 10seconds < 20 seconds > 20 subjects) FIG. 8 RU RU RU Reference Y (1) 5467 31 Reference Y + (2) 90 92 6 C-terminal Ala Reference Y + (3) 97 98 0L11V + V89L + C-terminal Ala Reference Y + (4) 98 98 0 L11V + T87A +V89L + C-terminal Ala Reference Y + (5) 96 98 0 L11V + V89L + T110K + C-terminal Ala Reference Y + (6) 98 98 0 L11V + V89L + T110Q + C- terminalAlaThree representative trivalent constructs were also tested against serumsamples obtained from SLE patients. The results are shown in Table Q.

TABLE Q testing of representative trivalent constructs against serumsamples obtained from HLE patients. The constructs (had, were indicated,a C- terminal alanine residue as well as, in each of the buildingblocks, the indicated mutations. Sample from healthy Mutation(s) Samplesobtained from SLE patients volunteer L11V T87A V89L T110K T110Q 114ASLE25 SLE37 SLE39 SLE41 NB13025-14 004-030-ABL-02 Average binding to[Reference X + C-terminal Ala] Reference X + 142 194 126 108 102 27C-terminal alanine Inhibition compared to average binding to [ReferenceX + C-terminal Ala] captured on HSA (%) Reference X + L11V + x x x 10095 100 88 100 0 V89L + C-terminal alanine Reference X + L11V + x x x x100 76 100 100 100 100 V89L + T110K + C-terminal alanine Reference X +L11V + x x x x 100 100 100 100 97 100 87A + V89L + C-terminal alanine

Example 7: Nanobodies and Nanobody-Constructs Against the Ion ChannelKv1.3

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstthe ion channel Kv.1.3.

The co-pending US provisional application U.S. Ser. No. 62/014,023(title: “Kv1.3 binding immunoglobulins”; assignee: Ablynx N.V.; filingdate: Jun. 18, 2014) as well as the subsequent filed US provisionalapplication of the same title filed on Mar. 16, 2015 (assignee: AblynxN.V.) inter alia describe immunoglobulin single variable domains (and inparticular Nanobodies) that are directed against the potassium selectivevoltage-gated ion channel Kv1.3, as well as proteins, polypeptides andother Nanobody-based constructs that comprise at least one such Nanobodyagainst Kv1.3.

The mutations described in the present application (optionally suitablycombined with a C-terminal extension as described in herein and/or in WO12/175741) may also be suitably applied to the Nanobodies, proteins,polypeptides and other Nanobody-based constructs against Kv1.3 that aredescribed in these two US provisional applications.

Thus, in one aspect, the invention relates to a VH domain that isdirected against Kv1.3 and that is as further described herein for theISVD's of the invention (i.e. comprising the amino acidresidues/mutations as described herein).

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against Kv1.3 describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain that is directed against Kv1.3, in which (i) position 112 is K orQ; or (ii) position 110 is K or Q and position 11 is V; or (iii)position 89 is T; or (iv) position 89 is L and position 110 is K or Q;or (v) position 11 is V and position 89 is L; or any suitablecombination of (i) to (v). In particular, in such VH domains againstKv1.3:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains against Kv1.3 referred to in this Example may inparticular have CDR's that are as described on pages 5-10 of U.S. Ser.No. 62/014,023 (including any preferred aspects/embodiments of suchCDRs) or sequence-optimized versions thereof, as described in the otherUS provisional application referred to above.

In particular, the VH domains against Kv 1.3 referred to in this Examplemay in particular have a combination of CDR1, CDR2 and CDR3 that ischosen from one of the combination of CDR 1, CDR2 and CDR3 that arelisted as preferred aspects in the list that bridges pages 9 and 10 ofU.S. Ser. No. 62/014,023.

In some preferred, but non-limiting aspect of the inventions:

-   -   in a VH domain of the invention: (i) CDR1 (according to Kabat)        is the sequence of SEQ ID NO:166 or an amino acid sequence that        has one or two amino acid differences with the sequence of SEQ        ID NO:166; (ii) CDR2 (according to Kabat) is the sequence of SEQ        ID NO:167 or an amino acid sequence that has one or two amino        acid differences with the sequence of SEQ ID NO:167; and (iii)        CDR3 (according to Kabat) is the sequence of SEQ ID NO:168 or an        amino acid sequence that has one or two amino acid differences        with the sequence of SEQ ID NO:168; and even more        preferably: (i) CDR1 (according to Kabat) is the sequence of SEQ        ID NO:166; (ii) CDR2 (according to Kabat) is the sequence of SEQ        ID NO:167; and (iii) CDR3 (according to Kabat) is the sequence        of SEQ ID NO:168; and/or    -   in a VH domain of the invention: (i) CDR1 (according to Abm) is        the sequence of SEQ ID NO:169 or an amino acid sequence that has        one or two amino acid differences with the sequence of SEQ ID        NO:169; (ii) CDR2 (according to Abm) is the sequence of SEQ ID        NO:170 or an amino acid sequence that has one or two amino acid        differences with the sequence of SEQ ID NO:170; and (iii) CDR3        (according to Abm) is the sequence of SEQ ID NO:171 or an amino        acid sequence that has one or two amino acid differences with        the sequence of SEQ ID NO:171; and even more preferably: (i)        CDR1 (according to Abm) is the sequence of SEQ ID NO:169; (ii)        CDR2 (according to Abm) is the sequence of SEQ ID NO:170;        and (iii) CDR3 (according to Abm) is the sequence of SEQ ID        NO:178.

The VH domains of the invention against Kv1.3 may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against Kv1.3 or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

For example and without limitation, the VH domains of the inventionagainst Kv1.3 may be one of the sequences listed in Table A-1 of U.S.Ser. No. 62/014,023 (SEQ ID NO's: 1 to 123 in U.S. Ser. No. 62/014,023)or one of the sequences of listed in Table A-1 of US provisionalapplication entitled “Kv1.3 binding immunoglobulins” (assignee: AblynxN.V.; filing date: Mar. 16, 2015) (SEQ ID NO's: 1 to 123, 495, 498 to513 or 523 to 540 in said US provisional; and in particular the sequenceof SEQ ID NO: 495), but suitably with the mutations/specific amino acidresidues described herein for the ISVDs of the invention, and optionallysuitably with a C-terminal extension.

In one specific aspect, a Nanobody of the invention against Kv1.3 is avariant of the Nanobody of SEQ ID NO:137 (with at least 90% sequenceidentity with SEQ ID NO:137), in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against Kv1.3 and of compounds of the invention comprisingthe same are listed in FIG. 9A (monovalent Nanobodies: SEQ ID NO's: 138to 155) and FIG. 9B (trivalent bispecific half-life extended constructs:SEQ ID NO's: 156 to 164). Compounds of the invention that comprise oressentially consist of at least one (such as one, two or three)anti-Kv1.3 Nanobody chosen from the anti-Kv1.3 Nanobodies of SEQ IDNO's: 138 to 155 form a further aspect of the invention. Also, each ofthe compounds of SEQ ID NO's: 156 to 164 forms a further aspect of theinvention. In one specific aspect, such compounds contain two suchNanobodies of the invention against Kv1.3 and one Nanobody against humanserum albumin (which is preferably also a Nanobody of the invention).Also, again, such a construct may contain suitable linkers and aC-terminal extension.

The monovalent anti-Kv1.3 Nanobodies of SEQ ID NO's: 138 to 155 weregenerated by introducing the L11V and V89L mutations of the inventioninto the starting sequence of SEQ ID NO: 137 (reference). In addition,different combinations of humanizing (or other sequence-optimizing)mutations were introduced, such as E1D, A14P, G19R, M53A or M53Q orM53Y, T62S, A74S, K83R, S94G and/or T97E). The specific mutationsintroduced in each of the sequences of SEQ ID NO: 138 to 155 is given inFIG. 9A.

Some of the monovalent anti-Kv1.3 Nanobodies from FIG. 9A were alsoformatted as trivalent bispecific constructs comprising two Nanobodiesof the invention against Kv1.3 and one half-life extending Nanobody ofthe invention against human serum albumin (SEQ ID NO:109, also referredto as “ALB-82” in FIG. 9B). 35GS linkers were used, and all theconstructs have a C-terminal extension (a single C-terminal alanineresidue). The sequences of the resulting constructs are given in SEQ IDNO's 156 to 164. Of these, three constructs (SEQ ID NOs: 156, 157 and160) were tested for binding by pre-existing antibodies in samplesobtained from 47 diabetic human subjects and 90 healthy human subjects,using the general protocol described herein. The binding by pre-existingantibodies by samples from these two sets were compared to the referenceconstruct of SEQ ID NO:165, which is a corresponding trivalentbispecific construct based on the reference anti-Kv1.3 building block ofSEQ ID NO:137 and the serum albumin binder Alb-8 (SEQ ID NO:46), againcombined with a C-terminal alanine extension. The results are shown inFIG. 10 (samples from 47 diabetic patients) and FIG. 11 (samples from 90healthy volunteers). In each case, the constructs with the L11V and V89Lmutations of the invention showed reduced binding by pre-existingantibodies compared to the reference construct.

Example 8: VH Domains (and in Particular Nanobodies) Against IL-23, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstIL-23.

Such a VH domain of the invention against IL-23 will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to IL-23. In addition, such a VH domain of the invention againstIL-23 may also suitably have a C-terminal extension as described herein,in particular when said VH domain is monovalent or forms the C-terminalend of the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst IL-23 may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against IL-23 describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against IL-23, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against IL-23:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against IL-23 may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against IL-23 or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against IL-23comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 173(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 173; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 174 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 174; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 175 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 175.

More preferably, in a VH domain of the invention against IL-23 accordingto this aspect: (i) CDR1 is SEQ ID NO:173; (ii) CDR2 is SEQ ID NO: 174;and (iii) CDR3 is SEQ ID NO: 175.

In one specific aspect, a Nanobody of the invention against IL-23 is avariant of the Nanobody of SEQ ID NO:172 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO:172),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstIL-23 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:191 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 191; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 192 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 192; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 193 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 193.

More preferably, in a VH domain of the invention against IL-23 accordingto this aspect: (i) CDR1 is SEQ ID NO: 191; (ii) CDR2 is SEQ ID NO: 192;and (iii) CDR3 is SEQ ID NO: 193.

In one specific aspect, a Nanobody of the invention against IL-23 is avariant of the Nanobody of SEQ ID NO:172 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO:190),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of suchNanobodies of the invention against IL-23 are listed in FIG. 12A as SEQID NO's: 176 to 189 and FIG. 12B as SEQ ID NO's: 194 to 207,respectively; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against IL-23that comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 176 to 189 and/or 194 to207. Such compounds of the invention against IL-23 may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table Rbelow.

As described in for example WO 2009/068627, WO 2010/142534 andWO2011/135026, on particularly preferred class of Nanobody-basedcompounds against IL-23 are biparatopic compounds. Thus, in one aspectof the invention, a compound of the invention against IL-23 is abiparatopic construct that comprises one ISV that is either SEQ ID NO:172 or (preferably) an ISV of the invention that has been derived fromSEQ ID NO:172 (as described in this Example 8) and one ISV that iseither SEQ ID NO: 190 or (preferably) an ISV of the invention that hasbeen derived from SEQ ID NO: 190 (as described in this Example 8),provided that at least one (and preferably both) of these ISV's areISV's of the invention. Such biparatopic constructs may also behalf-life extended (i.e. by means of a serum albumin-binding ISV). Somespecific examples of such biparatopic constructs are given in SEQ ID NO:514 to 549.

Some specifically preferred examples of compounds of the inventionagainst IL-23 are given in FIG. 22 as SEQ ID NO's: 514 to 549; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst IL-23 and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 514 to 549. More generally,compounds of the invention against IL-23 may be as described in WO2009/068627, WO 2010/142534 and WO2011/135026, but comprising ISV's ofthe invention. They may also be used for the purposes described in WO2009/068627, WO 2010/142534 and WO2011/135026.

TABLE R Examples of compounds of the invention against IL-23.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [IL-23] [IL-23] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [IL-23]-X(n) [IL-23] = one of SEQ ID NO's:464 to 477 and/or 482 to 495 C-terminal extension Monovalent/[IL-23]-L₁-[SA] [IL-23] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[IL-23] [SA] = (see legend below) extended L₁ =(see legend below) Monovalent/ [IL-23]-L₁-[SA]-X(n) [IL-23] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[IL-23]-X(n)[SA] = (see legend below) extended/C- L₁ = (see legend below) terminalX(n) = (see legend below) extension Bivalent(²) [IL-23]-L₁-[IL-23] Atleast one [IL-23] present is = one of SEQ ID NO's: 464 to 477 and/or 482to 495 (⁴) L₁ = (see legend below) Bivalent/ [IL-23]-L₁-[IL-23]-X(n) Atleast one [IL-23] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482to 495 (⁴) extension(²) L₁ = (see legend below) X(n) = (see legendbelow) Bivalent/ [IL-23]-L₁-[IL-23]-L₂-[SA] At least one [IL-23] presentis = one of SEQ ID NO's: 464 to half-life [IL-23]-L₁-[SA]-L₂-[IL-23] 477and/or 482 to 495 (⁴) extended(²) [SA]-L₁-[IL-23]-L₂-[IL-23] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[IL-23]-L₁-[IL-23]-L₂-[SA]-X(n) At least one [IL-23] present is = one ofSEQ ID NO's: 464 to half-life [IL-23]-L₁-[SA]-L₂-[IL-23]-X(n) 477 and/or482 to 495 (⁴) extended/ [SA]-L₁-[IL-23]-L₂-[IL-23]-X(n) [SA] = (seelegend below) C-terminal L₁ = (see legend below) extension(²) L₂ = (seelegend below) X(n) = (see legend below) Bispecific (³) [IL-23]-L₁-[Nb]At least one [IL-23] present is = one of SEQ ID NO's: 464 to[Nb]-L₁-[IL-23] 477 and/or 482 to 495 (⁴) [IL-23]-L₁-[IL-23]-L₂-[Nb] L₁= (see legend below) [IL-23]-L₁-[Nb]-L₂-[IL-23] L₂ = (see legend below)[Nb]-L₁-[IL-23]-L₂-[IL-23] [Nb] = (see legend below) Bispecific/[IL-23]-L₁-[Nb]-X(n) At least one [IL-23] present is = one of SEQ IDNO's: 464 to C-terminal [Nb]-L₁-[IL-23]-X(n) 477 and/or 482 to 495 (⁴)extension (3) [IL-23]-L₁-[IL-23]-L₂-[Nb]-X(n) L₁ = (see legend below)[IL-23]-L₁-[Nb]-L₂-[IL-23]-X(n) L₂ = (see legend below)[Nb]-L₁-[IL-23]-L₂-[IL-23]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [IL-23]-L₁-[Nb]-L₂-[SA] At least one [IL-23]present is = one of SEQ ID NO's: 464 to half-life[IL-23]-L₁-[SA]-L₂-[Nb] 477 and/or 482 to 495 (⁴) extended (³)[Nb]-L₁-[SA]-L₂-[IL-23] [SA] = (see legend below)[Nb]-L₁-[IL-23]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[IL-23]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[IL-23] Bispecific/ [IL-23]-L₁-[IL-23]-L₂-[Nb]-L₃-[SA]At least one [IL-23] present is = one of SEQ ID NO's: 464 to half-life[Nb]-L₁-[IL-23]-L₂-[IL-23]-L₃-[SA] 477 and/or 482 to 495 (⁴) extended(³) [SA]-L₁-[IL-23]-L₂-[IL-23]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[IL-23]-L₂-[Nb]-L₃-[IL-23] L₁, L₂, L₃(see legend below)[IL-23]-L₁-[Nb]-L₂-[IL-23]-L₃-[SA] [Nb] = (see legend below) Bispecific/[IL-23]-L₁-[Nb]-L₂-[SA]-X(n) At least one [IL-23] present is = one ofSEQ ID NO's: 464 to half-life [IL-23]-L₁-[SA]-L₂-[Nb]-X(n) 477 and/or482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[IL-23]-X(n) [SA] = (see legendbelow) C-terminal [Nb]-L₁-[IL-23]-L₂-[SA]-X(n) L₁, L₂, L₃(see legendbelow) extension (³) [SA]-L₁-[IL-23]-L₂-[Nb]-X(n) X(n) = (see legendbelow) [SA]-L₁-[Nb]-L₂-[IL-23]-X(n) [Nb] = (see legend below)[IL-23]-L₁-[IL-23]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[IL-23]-L₂-[IL-23]-L₃-[SA]-X(n)[SA]-L₁-[IL-23]-L₂-[IL-23]-L₃-[Nb]-X(n)[SA]-L₁-[IL-23]-L₂-[Nb]-L₃-[IL-23]-X(n)[IL-23]-L₁-[Nb]-L₂-[IL-23]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD' s of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against IL-23. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstIL-23 (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against IL-23 and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against IL-23 present is an ISV of theinvention, and preferably all of the ISV's against IL-23 present in suchpolypeptide/construct are ISV's of the invention. Also, when a half-lifeextending ISV and/or an ISV against another therapeutic target ispresent in such polypeptide/construct, each of these (and preferably allof these) may also be (and preferably are) ISV's of the invention (²)All“bivalent” constructs in this Table may also be biparatopic, meaningthat they comprise at least two (such as two) ISV's against IL-23, whichare directed against different epitopes on IL-23. (³) As will be clearto the skilled person, other/further bispecific constructs than thoselisted can be made using the building blocks and linkers mentioned. (⁴)Preferably, each [IL-23] present is independently chosen from SEQ IDNO's: 464 to 477 and/or 482 to 495. Also, the [IL-23] present may be thesame or different; in a biparatopic polypeptide/construct they will bedirected against different epitopes on IL-23. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 9: VH Domains (and in Particular Nanobodies) Against OX40-L, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstOX40-L.

Such a VH domain of the invention against OX40-L will generallycomprise: (i) suitable framework sequences that suitably comprise theamino acid residues/mutations of the invention as described herein; aswell as (ii) CDR sequences that allow the VH domain of the invention tospecifically bind to OX40-L. In addition, such a VH domain of theinvention against OX40-L may also suitably have a C-terminal extensionas described herein, in particular when said VH domain is monovalent orforms the C-terminal end of the compound of the invention in which saidVH domain is present (again, as further described herein). Such VHdomains of the invention against OX40-L may further be as furtherdescribed herein, and may in particular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against OX40-L describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against OX40-L, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against OX40-L:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against OX40-L may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against OX40-L or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against OX40-Lcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 209(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 209; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 210 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 210; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 211 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 211.

More preferably, in a VH domain of the invention against OX40-Laccording to this aspect: (i) CDR1 is SEQ ID NO:209; (ii) CDR2 is SEQ IDNO: 210; and (iii) CDR3 is SEQ ID NO: 211.

In one specific aspect, a Nanobody of the invention against OX40-L is avariant of the Nanobody of SEQ ID NO:208 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO:208),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against OX40-L are listed in FIG. 13 as SEQ ID NO's: 212to 225; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against OX40-Lthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 212 to 225. Such compoundsof the invention against OX40-L may again be as further describedherein, and thus for example may comprise suitable linkers, may comprisea C-terminal extension as described herein, and may be half-lifeextended (for example because they comprise a Nanobody against humanserum albumin, such as (preferably) a Nanobody of the invention againsthuman serum albumin). Reference is made to Table S below.

Some specifically preferred examples of compounds of the inventionagainst OX40-L are given in FIG. 23 as SEQ ID NO's: 550 to 585; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst OX40-L and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 550 to 585. More generally,compounds of the invention against OX40-L may be as described in WO2011/073180, but comprising ISV's of the invention. They may also beused for the purposes described in WO 2011/073180.

TABLE S Examples of compounds of the invention against OX40-L.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [OX40-L] [OX40-L] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [OX40-L]-X(n) [OX40-L] = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 C-terminal extension Monovalent/[OX40-L]-L₁-[SA] [OX40-L] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[OX40-L] [SA] = (see legend below) extended L₁ =(see legend below) Monovalent/ [OX40-L]-L₁-[SA]-X(n) [OX40-L] = one ofSEQ ID NO's: 464 to 477 and/or 482 to 495 half-life[SA]-L₁-[OX40-L]-X(n) [SA] = (see legend below) extended/C- L₁ = (seelegend below) terminal X(n) = (see legend below) extension Bivalent(²)[OX40-L]-L₁-[OX40-L] At least one [OX40-L] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[OX40-L]-L₁-[OX40-L]-X(n) At least one [OX40-L] = one of SEQ ID NO's:464 to 477 C-terminal and/or 482 to 495 (⁴) extension(²) L₁ = (seelegend below) X(n) = (see legend below) Bivalent/[OX40-L]-L₁-[OX40-L]-L₂-[SA] At least one [OX40-L] present is = one ofSEQ ID NO's: 464 to half-life [OX40-L]-L₁-[SA]-L₂-[OX40-L] 477 and/or482 to 495 (⁴) extended(²) [SA]-L₁-[OX40-L]-L₂-[OX40-L] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[OX40-L]-L₁-[OX40-L]-L₂-[SA]-X(n) At least one [OX40-L] present is = oneof SEQ ID NO's: 464 to half-life [OX40-L]-L₁-[SA]-L₂-[OX40-L]-X(n) 477and/or 482 to 495 (⁴) extended/ [SA]-L₁-[OX40-L]-L₂-[OX40-L]-X(n) [SA] =(see legend below) C-terminal L₁ = (see legend below) extension(²) L₂ =(see legend below) X(n) = (see legend below) Bispecific (³)[OX40-L]-L₁-[Nb] At least one [OX40-L] present is = one of SEQ ID NO's:464 to [Nb]-L₁-[OX40-L] 477 and/or 482 to 495 (⁴)[OX40-L]-L₁-[OX40-L]-L₂-[Nb] L₁ = (see legend below)[OX40-L]-L₁-[Nb]-L₂-[OX40-L] L₂ = (see legend below)[Nb]-L₁-[OX40-L]-L₂-[OX40-L] [Nb] = (see legend below) Bispecific/[OX40-L]-L₁-[Nb]-X(n) At least one [OX40-L] present is = one of SEQ IDNO's: 464 to C-terminal [Nb]-L₁-[OX40-L]-X(n) 477 and/or 482 to 495 (⁴)extension (3) [OX40-L]-L₁-[OX40-L]-L₂-[Nb]-X(n) L₁ = (see legend below)[OX40-L]-L₁-[Nb]-L₂-[OX40-L]-X(n) L₂ = (see legend below)[Nb]-L₁-[OX40-L]-L₂-[OX40-L]-X(n) X(m) = (see legend below) [Nb] = (seelegend below) Bispecific/ [OX40-L]-L₁-[Nb]-L₂-[SA] At least one [OX40-L]present is = one of SEQ ID NO's: 464 to half-life[OX40-L]-L₁-[SA]-L₂-[Nb] 477 and/or 482 to 495 (⁴) extended (³)[Nb]-L₁-[SA]-L₂-[OX40-L] [SA] = (see legend below)[Nb]-L₁-[OX40-L]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[OX40-L]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[OX40-L] Bispecific/[OX40-L]-L₁-[OX40-L]-L₂-[Nb]-L₃-[SA] At least one [OX40-L] present is =one of SEQ ID NO's: 464 to half-life[Nb]-L₁-[OX40-L]-L₂-[OX40-L]-L₃-[SA] 477 and/or 482 to 495 (⁴) extended(³) [SA]-L₁-[OX40-L]-L₂-[OX40-L]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[OX40-L]-L₂-[Nb]-L₃-[OX40-L] L₁, L₂, L₃(see legend below)[OX40-L]-L₁-[Nb]-L₂-[OX40-L]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [OX40-L]-L₁-[Nb]-L₂-[SA]-X(n) At least one [OX40-L] presentis = one of SEQ ID NO's: 464 to half-life [OX40-L]-L₁-[SA]-L₂-[Nb]-X(n)477 and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[OX40-L]-X(n) [SA] =(see legend below) C-terminal [Nb]-L₁-[OX40-L]-L₂-[SA]-X(n) L₁, L₂,L₃(see legend below) extension (³) [SA]-L₁-[OX40-L]-L₂-[Nb]-X(n) X(n) =(see legend below) [SA]-L₁-[Nb]-L₂-[OX40-L]-X(n) [Nb] = (see legendbelow) [OX40-L]-L₁-[OX40-L]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[OX40-L]-L₂-[OX40-L]-L₃-[SA]-X(n)[SA]-L₁-[OX40-L]-L₂-[OX40-L]-L₃-[Nb]-X(n)[SA]-L₁-[OX40-L]-L₂-[Nb]-L₃-[OX40-L]-X(n)[OX40-L]-L₁-[Nb]-L₂-[OX40-L]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against OX40-L. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstOX40-L (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against OX40-L and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against OX40-L present is an ISV of theinvention, and preferably all of the ISV's against OX40-L present insuch polypeptide/construct are ISV's of the invention. Also, when ahalf-life extending ISV and/or an ISV against another therapeutic targetis present in such polypeptide/construct, each of these (and preferablyall of these) may also be (and preferably are) ISV's of the invention(²)All “bivalent” constructs in this Table may also be biparatopic,meaning that they comprise at least two (such as two) ISV's againstOX40-L, which are directed against different epitopes on OX40-L. (³) Aswill be clear to the skilled person, other/further bispecific constructsthan those listed can be made using the building blocks and linkersmentioned. (⁴) Preferably, each [OX40-L] present is independently chosenfrom SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [OX40-L]present may be the same or different; in a biparatopicpolypeptide/construct they will be directed against different epitopeson OX40-L. (⁵) Each of the polypeptides/constructs mentioned to in thiscolumn by means of reference to a SEQ ID forms an individual specificaspect of the invention.

Example 10: VH Domains (and in Particular Nanobodies) Against IgE, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstIgE.

Such a VH domain of the invention against IgE will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to IgE. In addition, such a VH domain of the invention against IgEmay also suitably have a C-terminal extension as described herein, inparticular when said VH domain is monovalent or forms the C-terminal endof the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst IgE may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against IgE described inthis Example) or compound comprising the same is said to be “accordingto the invention” or “as further described herein”, the preferredaspects/embodiments and preferences that are generally described hereinfor the ISVD's or compounds of the invention also specifically apply tosaid specific ISVD or compound, respectively, unless explicitlyindicated otherwise or unless the specific technical context requiresotherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against IgE, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against IgE:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against IgE may further be as describedherein and may again in particular be an ISVD (and more in particular aNanobody) against IgE or a protein, polypeptide or other compound orconstruct that comprises as least one such ISVD. Such a protein,polypeptide or other compound or construct may also be as furtherdescribed herein, and may for example have an increased half-life (i.e.as described herein, e.g. a half-life—expressed as t½ beta—in humansubjects of in human subjects of at least 1 day, preferably at least 3days, more preferably at least 7 days, such as at least 10 days), andfor this purpose may for example comprise a serum-albumin bindingNanobody, which may also be a serum-albumin binding Nanobody of theinvention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against IgEcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 227(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 227; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 228 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 228; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 229 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 229.

More preferably, in a VH domain of the invention against IgE accordingto this aspect: (i) CDR1 is SEQ ID NO: 227; (ii) CDR2 is SEQ ID NO: 228;and (iii) CDR3 is SEQ ID NO: 229.

In one specific aspect, a Nanobody of the invention against IgE is avariant of the Nanobody of SEQ ID NO: 226 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 226),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against IgE are listed in FIG. 14 as SEQ ID NO's: 230 to243; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against IgEthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 230 to 243. Such compoundsof the invention against IgE may again be as further described herein,and thus for example may comprise suitable linkers, may comprise aC-terminal extension as described herein, and may be half-life extended(for example because they comprise a Nanobody against human serumalbumin, such as (preferably) a Nanobody of the invention against humanserum albumin). Reference is made to Table T below.

Some specifically preferred examples of compounds of the inventionagainst IgE are given in FIG. 24 as SEQ ID NO's: 586 to 594; and each ofthese compounds form a further aspect of the invention. Thus, in anotheraspect, the invention relates to a polypeptide that is directed againstIgE and that has an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 586 to 594.

More generally, compounds of the invention against IgE may be asdescribed in WO 2012/175740 and the relevant parts of WO2012/175400, butcomprising ISV's of the invention. They may also be used for thepurposes described in WO 2012/175740.

TABLE T Examples of compounds of the invention against IgE. Polypeptide/construct(¹) General formula ISV building blocks and linkers Monovalent[IgE] [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495Monovalent/ [IgE]-X(n) [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482to 495 C-terminal extension Monovalent/ [IgE]-L₁-[SA] [IgE] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[IgE] [SA] =(see legend below) extended L₁ = (see legend below) Monovalent/[IgE]-L₁-[SA]-X(n) [IgE] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[IgE]-X(n) [SA] = (see legend below) extended/C-L₁ = (see legend below) terminal X(n) = (see legend below) extensionBivalent(²) [IgE]-L₁-[IgE] At least one [IgE] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[IgE]-L₁-[IgE]-X(n) At least one [IgE] = one of SEQ ID NO's: 464 to 477and/or C-terminal 482 to 495 (⁴) extension(²) L₁ = (see legend below)X(n) = (see legend below) Bivalent/ [IgE]-L₁-[IgE]-L₂-[SA] At least one[IgE] present is = one of SEQ ID NO's: 464 to half-life[IgE]-L₁-[SA]-L₂-[IgE] 477 and/or 482 to 495 (⁴) extended(²)[SA]-L₁-[IgE]-L₂-[IgE] [SA] = (see legend below) L₁ = (see legend below)L₂ = (see legend below) Bivalent/ [IgE]-L₁-[IgE]-L₂-[SA]-X(n) At leastone [IgE] present is = one of SEQ ID NO's: 464 to half-life[IgE]-L₁-[SA]-L₂-[IgE]-X(n) 477 and/or 482 to 495 (⁴) extended/[SA]-L₁-[IgE]-L₂-[IgE]-X(n) [SA] = (see legend below) C-terminal L₁ =(see legend below) extension(²) L₂ = (see legend below) X(n) = (seelegend below) Bispecific (³) [IgE]-L₁-[Nb] At least one [IgE] present is= one of SEQ ID NO's: 464 to [Nb]-L₁-[IgE] 477 and/or 482 to 495 (⁴)[IgE]-L₁-[IgE]-L₂-[Nb] L₁ = (see legend below) [IgE]-L₁-[Nb]-L₂-[IgE] L₂= (see legend below) [Nb]-L₁-[IgE]-L₂-[IgE] [Nb] = (see legend below)Bispecific/ [IgE]-L₁-[Nb]-X(n) At least one [IgE] present is = one ofSEQ ID NO's: 464 to C-terminal [Nb]-L₁-[IgE]-X(n) 477 and/or 482 to 495(⁴) extension (3) [IgE]-L₁-[IgE]-L₂-[Nb]-X(n) L₁ = (see legend below)[IgE]-L₁-[Nb]-L₂-[IgE]-X(n) L₂ = (see legend below)[Nb]-L₁-[IgE]-L₂-[IgE]-X(n) X(n) = (see legend below) [Nb] = (see legendbelow) Bispecific/ [IgE]-L₁-[Nb]-L₂-[SA] At least one [IgE] present is =one of SEQ ID NO's: 464 to half-life [IgE]-L₁-[SA]-L₂-[Nb] 477 and/or482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[IgE] [SA] = (see legendbelow) [Nb]-L₁-[IgE]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[IgE]-L₂-[Nb] [Nb] = (see legend below) [SA]-L₁-[Nb]-L₂-[IgE]Bispecific/ [IgE]-L₁-[IgE]-L₂-[Nb]-L₃-[SA] At least one [IgE] present is= one of SEQ ID NO's: 464 to half-life [Nb]-L₁-[IgE]-L₂-[IgE]-L₃-[SA]477 and/or 482 to 495 (⁴) extended (³) [SA]-L₁-[IgE]-L₂-[IgE]-L₃-[Nb][SA] = (see legend below) [SA]-L₁-[IgE]-L₂-[Nb]-L₃-[IgE] L₁, L₂, L₃(seelegend below) [IgE]-L₁-[Nb]-L₂-[IgE]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [IgE]-L₁-[Nb]-L₂-[SA]-X(n) At least one [IgE] present is =one of SEQ ID NO's: 464 to half-life [IgE]-L₁-[SA]-L₂-[Nb]-X(n) 477and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[IgE]-X(n) [SA] = (seelegend below) C-terminal [Nb]-L₁-[IgE]-L₂-[SA]-X(n) L₁, L₂, L₃(seelegend below) extension (³) [SA]-L₁-[IgE]-L₂-[Nb]-X(n) X(n) = (seelegend below) [SA]-L₁-[Nb]-L₂-[IgE]-X(n) [Nb] = (see legend below)[IgE]-L₁-[IgE]-L₂-[Nb]-L₃-[SA]-X(n) [Nb]-L₁-[IgE]-L₂-[IgE]-L₃-[SA]-X(n)[SA]-L₁-[IgE]-L₂-[IgE]-L₃-[Nb]-X(n) [SA]-L₁-[IgE]-L₂-[Nb]-L₃-[IgE]-X(n)[IgE]-L₁-[Nb]-L₂-[IgE]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against IgE. These may further comprise a half-life extendingISV (such as an ISV against serum albumin). “Bivalent” generally refersto polypeptides/constructs comprising two ISV's against IgE (which maybe the same or different). These may again further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bispecific”generally refers to polypeptides/constructs comprising at least one(such as 1 or 2) ISV's against IgE and at least one (such as 1 or 2)other ISV against a therapeutic target. These may further comprise ahalf-life extending ISV (such as an ISV against serum albumin). In thepolypeptides/constructs described in this table, at least one of theISV's against IgE present is an ISV of the invention, and preferably allof the ISV's against IgE present in such polypeptide/construct are ISV'sof the invention. Also, when a half-life extending ISV and/or an ISVagainst another therapeutic target is present in suchpolypeptide/construct, each of these (and preferably all of these) mayalso be (and preferably are) ISV's of the invention (²)All “bivalent”constructs in this Table may also be biparatopic, meaning that theycomprise at least two (such as two) ISV's against IgE, which aredirected against different epitopes on IgE. (³) As will be clear to theskilled person, other/further bispecific constructs than those listedcan be made using the building blocks and linkers mentioned. (⁴)Preferably, each [IgE] present is independently chosen from SEQ ID NO's:464 to 477 and/or 482 to 495. Also, the [IgE] present may be the same ordifferent; in a biparatopic polypeptide/construct they will be directedagainst different epitopes on IgE. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 11: VH Domains (and in Particular Nanobodies) Against CXCR4, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstCXCR4.

Such a VH domain of the invention against CXCR4 will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to CXCR4. In addition, such a VH domain of the invention againstCXCR4 may also suitably have a C-terminal extension as described herein,in particular when said VH domain is monovalent or forms the C-terminalend of the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst CXCR4 may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against CXCR4 describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against CXCR4, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against CXCR4:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against CXCR4 may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against CXCR4 or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against CXCR4comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 245(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 245; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 246 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 246; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 247 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 247.

More preferably, in a VH domain of the invention against CXCR4 accordingto this aspect: (i) CDR1 is SEQ ID NO: 245; (ii) CDR2 is SEQ ID NO: 246;and (iii) CDR3 is SEQ ID NO: 247.

In one specific aspect, a Nanobody of the invention against CXCR4 is avariant of the Nanobody of SEQ ID NO: 244 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 244),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstCXCR4 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:263 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 263; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 264 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 264; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 265 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 265.

More preferably, in a VH domain of the invention against CXCR-4according to this aspect: (i) CDR1 is SEQ ID NO: 263; (ii) CDR2 is SEQID NO: 264; and (iii) CDR3 is SEQ ID NO: 265.

In one specific aspect, a Nanobody of the invention against IL-23 is avariant of the Nanobody of SEQ ID NO: 262 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 262),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against CXCR4 are listed in FIG. 15A as SEQ ID NO's: 248to 261 and in FIG. 15B as SEQ ID NO's: 266 to 279; and each of theseNanobodies form a further aspect of the invention.

The invention also relates to a compound of the invention against CXCR4that comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 248 to 261 and/or 266 to279. Such compounds of the invention against CXCR4 may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table Ubelow.

As described in for example WO 2009/138519, WO 2011/042398 and WO2011/161266, one particularly preferred class of Nanobody-basedcompounds against CXCR4 are biparatopic compounds. Thus, in one aspectof the invention, a compound of the invention against CXCR4 is abiparatopic construct that comprises one ISV that is either SEQ ID NO:244 or (preferably) an ISV of the invention that has been derived fromSEQ ID NO: 244 (as described in this Example 11) and one ISV that iseither SEQ ID NO: 262 or (preferably) an ISV of the invention that hasbeen derived from SEQ ID NO: 262 (as described in this Example 11),provided that at least one (and preferably both) of these ISV's areISV's of the invention. Such biparatopic constructs may also behalf-life extended (i.e. by means of a serum albumin-binding ISV). Somespecific examples of such biparatopic constructs are given in SEQ ID NO:595 to 603.

Some specifically preferred examples of compounds of the inventionagainst CXCR-4 are given in FIG. 25 as SEQ ID NO's: 595 to 603; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst CXCR-4 and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 595 to 603. More generally,compounds of the invention against CXCR-4 may be as described in WO2009/138519, WO 2011/042398 and WO 2011/161266WO 2011/144749, butcomprising ISV's of the invention. They may also be used for thepurposes described in WO 2009/138519, WO 2011/042398 and WO 2011/161266.

TABLE U Examples of compounds of the invention against CXCR-4.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [CXCR-4] [CXCR-4] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [CXCR-4]-X(n) [CXCR-4] = one of SEQ IDNO's: 464 to 477 and/or 482 to C-terminal 495 extension Monovalent/[CXCR-4]-L₁-[SA] [CXCR-4] = one of SEQ ID NO's: 464 to 477 and/or 482 tohalf-life [SA]-L₁-[CXCR-4] 495 extended [SA] = (see legend below) L₁ =(see legend below) Monovalent/ [CXCR-4]-L₁-[SA]-X(n) [CXCR-4] = one ofSEQ ID NO's: 464 to 477 and/or 482 to half-life [SA]-L₁-[CXCR-4]-X(n)495 extended/C- [SA] = (see legend below) terminal L₁ = (see legendbelow) extension X(n) = (see legend below) Bivalent(²)[CXCR-4]-L₁-[CXCR-4] At least one [CXCR-4] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[CXCR-4]-L₁-[CXCR-4]-X(n) At least one [CXCR-4] = one of SEQ ID NO's:464 to 477 C-terminal and/or 482 to 495 (⁴) extension(²) L₁ = (seelegend below) X(n) = (see legend below) Bivalent/[CXCR-4]-L₁-[CXCR-4]-L₂-[SA] At least one [CXCR-4] present is = one ofSEQ ID NO's: 464 half-life [CXCR-4]-L₁-[SA]-L₂-[CXCR-4] to 477 and/or482 to 495 (⁴) extended(²) [SA]-L₁-[CXCR-4]-L₂-[CXCR-4] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[CXCR-4]-L₁-[CXCR-4]-L₂-[SA]-X(n) At least one [CXCR-4] present is = oneof SEQ ID NO's: 464 half-life [CXCR-4]-L₁-[SA]-L₂-[CXCR-4]-X(n) to 477and/or 482 to 495 (⁴) extended/ [SA]-L₁-[CXCR-4]-L₂-[CXCR-4]-X(n) [SA] =(see legend below) C-terminal L₁ = (see legend below) extension(²) L₂ =(see legend below) X(n) = (see legend below) Bispecific (³)[CXCR-4]-L₁-[Nb] At least one [CXCR-4] present is = one of SEQ ID NO's:464 [Nb]-L₁-[CXCR-4] to 477 and/or 482 to 495 (⁴)[CXCR-4]-L₁-[CXCR-4]-L₂-[Nb] L₁ = (see legend below)[CXCR-4]-L₁-[Nb]-L₂-[CXCR-4] L₂ = (see legend below)[Nb]-L₁-[CXCR-4]-L₂-[CXCR-4] [Nb] = (see legend below) Bispecific/[CXCR-4]-L₁-[Nb]-X(n) At least one [CXCR-4] present is = one of SEQ IDNO's: 464 C-terminal [Nb]-L₁-[CXCR-4]-X(n) to 477 and/or 482 to 495 (⁴)extension (3) [CXCR-4]-L₁-[CXCR-4]-L₂-[Nb]-X(n) L₁ = (see legend below)[CXCR-4]-L₁-[Nb]-L₂-[CXCR-4]-X(n) L₂ = (see legend below)[Nb]-L₁-[CXCR-4]-L₂-[CXCR-4]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [CXCR-4]-L₁-[Nb]-L₂-[SA] At least one [CXCR-4]present is = one of SEQ ID NO's: 464 half-life [CXCR-4]-L₁-[SA]-L₂-[Nb]to 477 and/or 482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[CXCR-4] [SA]= (see legend below) [Nb]-L₁-[CXCR-4]-L₂-[SA] L₁, L₂, L₃(see legendbelow) [SA]-L₁-[CXCR-4]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[CXCR-4] Bispecific/[CXCR-4]-L₁-[CXCR-4]-L₂-[Nb]-L₃-[SA] At least one [CXCR-4] present is =one of SEQ ID NO's: 464 half-life [Nb]-L₁-[CXCR-4]-L₂-[CXCR-4]-L₃-[SA]to 477 and/or 482 to 495 (⁴) extended (³)[SA]-L₁-[CXCR-4]-L₂-[CXCR-4]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[CXCR-4]-L₂-[Nb]-L₃-[CXCR-4] L₁, L₂, L₃(see legend below)[CXCR-4]-L₁-[Nb]-L₂-[CXCR-4]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [CXCR-4]-L₁-[Nb]-L₂-[SA]-X(n) At least one [CXCR-4] presentis = one of SEQ ID NO's: 464 half-life [CXCR-4]-L₁-[SA]-L₂-[Nb]-X(n) to477 and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[CXCR-4]-X(n) [SA] =(see legend below) C-terminal [Nb]-L₁-[CXCR-4]-L₂-[SA]-X(n) L₁, L₂,L₃(see legend below) extension (³) [SA]-L₁-[CXCR-4]-L₂-[Nb]-X(n) X(n) =(see legend below) [SA]-L₁-[Nb]-L₂-[CXCR-4]-X(n) [Nb] = (see legendbelow) [CXCR-4]-L₁-[CXCR-4]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[CXCR-4]-L₂-[CXCR-4]-L₃-[SA]-X(n)[SA]-L₁-[CXCR-4]-L₂-[CXCR-4]-L₃-[Nb]-X(n)[SA]-L₁-[CXCR-4]-L₂-[Nb]-L₃-[CXCR-4]-X(n)[CXCR-4]-L₁-[Nb]-L₂-[CXCR-4]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against CXCR-4. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstCXCR-4 (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against CXCR-4 and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against CXCR-4 present is an ISV of theinvention, and preferably all of the ISV's against CXCR-4 present insuch polypeptide/construct are ISV's of the invention. Also, when ahalf-life extending ISV and/or an ISV against another therapeutic targetis present in such polypeptide/construct, each of these (and preferablyall of these) may also be (and preferably are) ISV's of the invention(²)All “bivalent” constructs in this Table may also be biparatopic,meaning that they comprise at least two (such as two) ISV's againstCXCR-4, which are directed against different epitopes on CXCR-4. (³) Aswill be clear to the skilled person, other/further bispecific constructsthan those listed can be made using the building blocks and linkersmentioned. (⁴) Preferably, each [CXCR-4] present is independently chosenfrom SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [CXCR-4]present may be the same or different; in a biparatopicpolypeptide/construct they will be directed against different epitopeson CXCR-4. (⁵) Each of the polypeptides/constructs mentioned to in thiscolumn by means of reference to a SEQ ID forms an individual specificaspect of the invention.

Example 12: VH Domains (and in Particular Nanobodies) Against HER-3, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstHER-3.

Such a VH domain of the invention against HER-3 will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to HER-3. In addition, such a VH domain of the invention againstHER-3 may also suitably have a C-terminal extension as described herein,in particular when said VH domain is monovalent or forms the C-terminalend of the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst HER-3 may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against HER-3 describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against HER-3, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against HER-3:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against HER-3 may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against HER-3 or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against HER-3comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 281(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 281; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 282 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 282; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 283 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 283.

More preferably, in a VH domain of the invention against HER-3 accordingto this aspect: (i) CDR1 is SEQ ID NO: 281; (ii) CDR2 is SEQ ID NO: 282;and (iii) CDR3 is SEQ ID NO: 283.

In one specific aspect, a Nanobody of the invention against HER-3 is avariant of the Nanobody of SEQ ID NO: 280 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 280),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstHER-3 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:299 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 299; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 300 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 300; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 301 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 301.

More preferably, in a VH domain of the invention against HER-3 accordingto this aspect: (i) CDR1 is SEQ ID NO: 299; (ii) CDR2 is SEQ ID NO: 300;and (iii) CDR3 is SEQ ID NO: 301.

In one specific aspect, a Nanobody of the invention against HER-3 is avariant of the Nanobody of SEQ ID NO: 298 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 298),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against HER-3 are listed in FIG. 16A as SEQ ID NO's: 284to 297 and FIG. 16B as SEQ ID NO's: 302 to 315, respectively; and eachof these Nanobodies form a further aspect of the invention.

The invention also relates to a compound of the invention against HER-3that comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 284 to 297 and/or 302 to315. Such compounds of the invention against HER-3 may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table Vbelow.

As described in for example WO 2011/144749, one particularly preferredclass of Nanobody-based compounds against HER-3 are biparatopiccompounds. Thus, in one aspect of the invention, a compound of theinvention against HER-3 is a biparatopic construct that comprises oneISV that is either SEQ ID NO: 280 or (preferably) an ISV of theinvention that has been derived from SEQ ID NO: 280 (as described inthis Example 12) and one ISV that is either SEQ ID NO: 298 or(preferably) an ISV of the invention that has been derived from SEQ IDNO: 298 (as described in this Example 12), provided that at least one(and preferably both) of these ISV's are ISV's of the invention. Suchbiparatopic constructs may also be half-life extended (i.e. by means ofa serum albumin-binding ISV). Some specific examples of such biparatopicconstructs are given in SEQ ID NO: 604 to 639.

Some specifically preferred examples of compounds of the inventionagainst HER-3 are given in FIG. 26 as SEQ ID NO's: 604 to 639; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst HER-3 and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 604 to 639. More generally,compounds of the invention against HER-3 may be as described in WO2011/144749, but comprising ISV's of the invention. They may also beused for the purposes described in WO 2011/144749.

TABLE V Examples of compounds of the invention against HER-3.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [HER-3] [HER-3] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [HER-3]-X(n) [HER-3] = one of SEQ ID NO's:464 to 477 and/or 482 to 495 C-terminal extension Monovalent/[HER-3]-L₁-[SA] [HER-3] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[HER-3] [SA] = (see legend below) extended L₁ =(see legend below) Monovalent/ [HER-3]-L₁-[SA]-X(n) [HER-3] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[HER-3]-X(n)[SA] = (see legend below) extended/C- L₁ = (see legend below) terminalX(n) = (see legend below) extension Bivalent(²) [HER-3]-L₁-[HER-3] Atleast one [HER-3] present is = one of SEQ ID NO's: 464 to 477 and/or 482to 495 (⁴) L₁ = (see legend below) Bivalent/ [HER-3]-L₁-[HER-3]-X(n) Atleast one [HER-3] = one of SEQ ID NO's: 464 to 477 C-terminal and/or 482to 495 (⁴) extension(²) L₁ = (see legend below) X(n) = (see legendbelow) Bivalent/ [HER-3]-L₁-[HER-3]-L₂-[SA] At least one [HER-3] presentis = one of SEQ ID NO's: 464 to half-life [HER-3]-L₁-[SA]-L₂-[HER-3] 477and/or 482 to 495 (⁴) extended(²) [SA]-L₁-[HER-3]-L₂-[HER-3] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[HER-3]-L₁-[HER-3]-L₂-[SA]-X(n) At least one [HER-3] present is = one ofSEQ ID NO's: 464 to half-life [HER-3]-L₁-[SA]-L₂-[HER-3]-X(n) 477 and/or482 to 495 (⁴) extended/ [SA]-L₁-[HER-3]-L₂-[HER-3]-X(n) [SA] = (seelegend below) C-terminal L₁ = (see legend below) extension(²) L₂ = (seelegend below) X(n) = (see legend below) Bispecific (³) [HER-3]-L₁-[Nb]At least one [HER-3] present is = one of SEQ ID NO's: 464 to[Nb]-L₁-[HER-3] 477 and/or 482 to 495 (⁴) [HER-3]-L₁-[HER-3]-L₂-[Nb] L₁= (see legend below) [HER-3]-L₁-[Nb]-L₂-[HER-3] L₂ = (see legend below)[Nb]-L₁-[HER-3]-L₂-[HER-3] [Nb] = (see legend below) Bispecific/[HER-3]-L₁-[Nb]-X(n) At least one [HER-3] present is = one of SEQ IDNO's: 464 to C-terminal [Nb]-L₁-[HER-3]-X(n) 477 and/or 482 to 495 (⁴)extension (3) [HER-3]-L₁-[HER-3]-L₂-[Nb]-X(n) L₁ = (see legend below)[HER-3]-L₁-[Nb]-L₂-[HER-3]-X(n) L₂= (see legend below)[Nb]-L₁-[HER-3]-L₂-[HER-3]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [HER-3]-L₁-[Nb]-L₂-[SA] At least one [HER-3]present is = one of SEQ ID NO's: 464 to half-life[HER-3]-L₁-[SA]-L₂-[Nb] 477 and/or 482 to 495 (⁴) extended (³)[Nb]-L₁-[SA]-L₂-[HER-3] [SA] = (see legend below)[Nb]-L₁-[HER-3]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[HER-3]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[HER-3] Bispecific/ [HER-3]-L₁-[HER-3]-L₂-[Nb]-L₃-[SA]At least one [HER-3] present is = one of SEQ ID NO's: 464 to half-life[Nb]-L₁-[HER-3]-L₂-[HER-3]-L₃-[SA] 477 and/or 482 to 495 (⁴) extended(³) [SA]-L₁-[HER-3]-L₂-[HER-3]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[HER-3]-L₂-[Nb]-L₃-[HER-3] L₁, L₂, L₃(see legend below)[HER-3]-L₁-[Nb]-L₂-[HER-3]-L₃-[SA] [Nb] = (see legend below) Bispecific/[HER-3]-L₁-[Nb]-L₂-[SA]-X(n) At least one [HER-3] present is = one ofSEQ ID NO's: 464 to half-life [HER-3]-L₁-[SA]-L₂-[Nb]-X(n) 477 and/or482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[HER-3]-X(n) [SA] = (see legendbelow) C-terminal [Nb]-L₁-[HER-3]-L₂-[SA]-X(n) L₁, L₂, L₃(see legendbelow) extension (³) [SA]-L₁-[HER-3]-L₂-[Nb]-X(n) X(n) = (see legendbelow) [SA]-L₁-[Nb]-L₂-[HER-3]-X(n) [Nb] = (see legend below)[HER-3]-L₁-[HER-3]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[HER-3]-L₂-[HER-3]-L₃-[SA]-X(n)[SA]-L₁-[HER-3]-L₂-[HER-3]-L₃-[Nb]-X(n)[SA]-L₁-[HER-3]-L₂-[Nb]-L₃-[HER-3]-X(n)[HER-3]-L₁-[Nb]-L₂-[HER-3]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD' s of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against HER-3. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstHER-3 (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against HER-3 and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against HER-3 present is an ISV of theinvention, and preferably all of the ISV's against HER-3 present in suchpolypeptide/construct are ISV's of the invention. Also, when a half-lifeextending ISV and/or an ISV against another therapeutic target ispresent in such polypeptide/construct, each of these (and preferably allof these) may also be (and preferably are) ISV's of the invention (²)All“bivalent” constructs in this Table may also be biparatopic, meaningthat they comprise at least two (such as two) ISV's against HER-3, whichare directed against different epitopes on HER-3. (³) As will be clearto the skilled person, other/further bispecific constructs than thoselisted can be made using the building blocks and linkers mentioned. (⁴)Preferably, each present is independently chosen from SEQ ID NO's: 464to 477 and/or 482 to 495. Also, the present may be the same ordifferent; in a biparatopic polypeptide/construct they will be directedagainst different epitopes on HER-3. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 13: VH Domains (and in Particular Nanobodies) Against TNF, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstTNF.

Such a VH domain of the invention against TNF will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to TNF. In addition, such a VH domain of the invention against TNFmay also suitably have a C-terminal extension as described herein, inparticular when said VH domain is monovalent or forms the C-terminal endof the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst TNF may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against TNF described inthis Example) or compound comprising the same is said to be “accordingto the invention” or “as further described herein”, the preferredaspects/embodiments and preferences that are generally described hereinfor the ISVD's or compounds of the invention also specifically apply tosaid specific ISVD or compound, respectively, unless explicitlyindicated otherwise or unless the specific technical context requiresotherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against TNF, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against TNF:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against TNF may further be as describedherein and may again in particular be an ISVD (and more in particular aNanobody) against TNF or a protein, polypeptide or other compound orconstruct that comprises as least one such ISVD. Such a protein,polypeptide or other compound or construct may also be as furtherdescribed herein, and may for example have an increased half-life (i.e.as described herein, e.g. a half-life—expressed as t½ beta—in humansubjects of in human subjects of at least 1 day, preferably at least 3days, more preferably at least 7 days, such as at least 10 days), andfor this purpose may for example comprise a serum-albumin bindingNanobody, which may also be a serum-albumin binding Nanobody of theinvention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against TNFcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 317(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 317; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 318 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 318; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 319 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 319.

More preferably, in a VH domain of the invention against TNF accordingto this aspect: (i) CDR1 is SEQ ID NO: 317; (ii) CDR2 is SEQ ID NO: 318;and (iii) CDR3 is SEQ ID NO: 319.

In one specific aspect, a Nanobody of the invention against TNF is avariant of the Nanobody of SEQ ID NO: 316 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 316),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against TNF are listed in FIG. 17A as SEQ ID NO's: 320 to333; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against TNFthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 320 to 333. Such compoundsof the invention against TNF may again be as further described herein,and thus for example may comprise suitable linkers, may comprise aC-terminal extension as described herein, and may be half-life extended(for example because they comprise a Nanobody against human serumalbumin, such as (preferably) a Nanobody of the invention against humanserum albumin). Reference is made to Table W below. Generally, as TNF isa multivalent target, compounds of the invention comprising two or threeanti-TNF ISV's (and linkers of suitable length, see WO 06/122786) arepreferred)

Some specifically preferred examples of compounds of the inventionagainst TNF are given in FIG. 27 as SEQ ID NO's: 640 to 675; and each ofthese compounds form a further aspect of the invention. Thus, in anotheraspect, the invention relates to a polypeptide that is directed againstTNF and that has an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 640 to 675. More generally, compounds of theinvention against TNF may be as described in WO 2006/122786, butcomprising ISV's of the invention. They may also be used for thepurposes described in WO 2006/122786.

TABLE W Examples of compounds of the invention against TNF. Polypeptide/construct(¹) General formula ISV building blocks and linkers Monovalent[TNF] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495Monovalent/ [TNF]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482to 495 C-terminal extension Monovalent/ [TNF]-L₁-[SA] [TNF] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[TNF] [SA] =(see legend below) extended L₁ = (see legend below) Monovalent/[TNF]-L₁-[SA]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[TNF]-X(n) [SA] = (see legend below) extended/C-L₁ = (see legend below) terminal X(n) = (see legend below) extensionBivalent(²) [TNF]-L₁-[TNF] At least one [TNF] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[TNF]-L₁-[TNF]-X(n) At least one [TNF] = one of SEQ ID NO's: 464 to 477and/or C-terminal 482 to 495 (⁴) extension(²) L₁ = (see legend below)X(n) = (see legend below) Bivalent/ [TNF]-L₁-[TNF]-L₂-[SA] At least one[TNF] present is = one of SEQ ID NO's: 464 to half-life[TNF]-L₁-[SA]-L₂-[TNF] 477 and/or 482 to 495 (⁴) extended(²)[SA]-L₁-[TNF]-L₂-[TNF] [SA] = (see legend below) L₁ = (see legend below)L₂ = (see legend below) Bivalent/ [TNF]-L₁-[TNF]-L₂-[SA]-X(n) At leastone [TNF] present is = one of SEQ ID NO's: 464 to half-life[TNF]-L₁-[SA]-L₂-[TNF]-X(n) 477 and/or 482 to 495 (⁴) extended/[SA]-L₁-[TNF]-L₂-[TNF]-X(n) [SA] = (see legend below) C-terminal L₁ =(see legend below) extension(²) L₂ = (see legend below) X(n) = (seelegend below) Bispecific (³) [TNF]-L₁-[Nb] At least one [TNF] present is= one of SEQ ID NO's: 464 to [Nb]-L₁-[TNF] 477 and/or 482 to 495 (⁴)[TNF]-L₁-[TNF]-L₂-[Nb] L₁ = (see legend below) [TNF]-L₁-[Nb]-L₂-[TNF] L₂= (see legend below) [Nb]-L₁-[TNF]-L₂-[TNF] [Nb] = (see legend below)Bispecific/ [TNF]-L₁-[Nb]-X(n) At least one [TNF] present is = one ofSEQ ID NO's: 464 to C-terminal [Nb]-L₁-[TNF]-X(n) 477 and/or 482 to 495(⁴) extension (3) [TNF]-L₁-[TNF]-L₂-[Nb]-X(n) L₁ = (see legend below)[TNF]-L₁-[Nb]-L₂-[TNF]-X(n) L₂ = (see legend below)[Nb]-L₁-[TNF]-L₂-[TNF]-X(n) X(n) = (see legend below) [Nb] = (see legendbelow) Bispecific/ [TNF]-L₁-[Nb]-L₂-[SA] At least one [TNF] present is =one of SEQ ID NO's: 464 to half-life [TNF]-L₁-[SA]-L₂-[Nb] 477 and/or482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[TNF] [SA] = (see legendbelow) [Nb]-L₁-[TNF]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[TNF]-L₂-[Nb] [Nb] = (see legend below) [SA]-L₁-[Nb]-L₂-[TNF]Bispecific/ [TNF]-L₁-[TNF]-L₂-[Nb]-L₃-[SA] At least one [TNF] present is= one of SEQ ID NO's: 464 to half-life [Nb]-L₁-[TNF]-L₂-[TNF]-L₃-[SA]477 and/or 482 to 495 (⁴) extended (³) [SA]-L₁-[TNF]-L₂-[TNF]-L₃-[Nb][SA] = (see legend below) [SA]-L₁-[TNF]-L₂-[Nb]-L₃-[TNF] L₁, L₂, L₃(seelegend below) [TNF]-L₁-[Nb]-L₂-[TNF]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [TNF]-L₁-[Nb]-L₂-[SA]-X(n) At least one [TNF] present is =one of SEQ ID NO's: 464 to half-life [TNF]-L₁-[SA]-L₂-[Nb]-X(n) 477and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[TNF]-X(n) [SA] = (seelegend below) C-terminal [Nb]-L₁-[TNF]-L₂-[SA]-X(n) L₁, L₂, L₃(seelegend below) extension (³) [SA]-L₁-[TNF]-L₂-[Nb]-X(n) X(n) = (seelegend below) [SA]-L₁-[Nb]-L₂-[TNF]-X(n) [Nb] = (see legend below)[TNF]-L₁-[TNF]-L₂-[Nb]-L₃-[SA]-X(n) [Nb]-L₁-[TNF]-L₂-[TNF]-L₃-[SA]-X(n)[SA]-L₁-[TNF]-L₂-[TNF]-L₃-[Nb]-X(n) [SA]-L₁-[TNF]-L₂-[Nb]-L₃-[TNF]-X(n)[TNF]-L₁-[Nb]-L₂-[TNF]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against TNF. These may further comprise a half-life extendingISV (such as an ISV against serum albumin). “Bivalent” generally refersto polypeptides/constructs comprising two ISV's against TNF (which maybe the same or different). These may again further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bispecific”generally refers to polypeptides/constructs comprising at least one(such as 1 or 2) ISV's against TNF and at least one (such as 1 or 2)other ISV against a therapeutic target. These may further comprise ahalf-life extending ISV (such as an ISV against serum albumin). In thepolypeptides/constructs described in this table, at least one of theISV's against TNF present is an ISV of the invention, and preferably allof the ISV's against TNF present in such polypeptide/construct are ISV'sof the invention. Also, when a half-life extending ISV and/or an ISVagainst another therapeutic target is present in suchpolypeptide/construct, each of these (and preferably all of these) mayalso be (and preferably are) ISV's of the invention (²)All “bivalent”constructs in this Table may also be biparatopic, meaning that theycomprise at least two (such as two) ISV's against TNF, which aredirected against different epitopes on TNF. (³) As will be clear to theskilled person, other/further bispecific constructs than those listedcan be made using the building blocks and linkers mentioned. (⁴)Preferably, each present is independently chosen from SEQ ID NO's: 464to 477 and/or 482 to 495. Also, the present may be the same ordifferent; in a biparatopic polypeptide/construct they will be directedagainst different epitopes on TNF. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 14: Further VH Domains (and in Particular Nanobodies) AgainstTNF, and Compounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstTNF.

Such a VH domain of the invention against TNF will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to TNF. In addition, such a VH domain of the invention against TNFmay also suitably have a C-terminal extension as described herein, inparticular when said VH domain is monovalent or forms the C-terminal endof the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst TNF may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against TNF described inthis Example) or compound comprising the same is said to be “accordingto the invention” or “as further described herein”, the preferredaspects/embodiments and preferences that are generally described hereinfor the ISVD's or compounds of the invention also specifically apply tosaid specific ISVD or compound, respectively, unless explicitlyindicated otherwise or unless the specific technical context requiresotherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against TNF, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against TNF:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against TNF may further be as describedherein and may again in particular be an ISVD (and more in particular aNanobody) against TNF or a protein, polypeptide or other compound orconstruct that comprises as least one such ISVD. Such a protein,polypeptide or other compound or construct may also be as furtherdescribed herein, and may for example have an increased half-life (i.e.as described herein, e.g. a half-life—expressed as t½ beta—in humansubjects of in human subjects of at least 1 day, preferably at least 3days, more preferably at least 7 days, such as at least 10 days), andfor this purpose may for example comprise a serum-albumin bindingNanobody, which may also be a serum-albumin binding Nanobody of theinvention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against TNFcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 335(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 335; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 336 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 336; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 337 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 337.

More preferably, in a VH domain of the invention against TNF accordingto this aspect: (i) CDR1 is SEQ ID NO: 335; (ii) CDR2 is SEQ ID NO: 336;and (iii) CDR3 is SEQ ID NO: 337.

In one specific aspect, a Nanobody of the invention against TNF is avariant of the Nanobody of SEQ ID NO: 334 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 334),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against TNF are listed in FIG. 17B as SEQ ID NO's: 338 to351; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against TNFthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 338 to 251. Such compoundsof the invention against TNF may again be as further described herein,and thus for example may comprise suitable linkers, may comprise aC-terminal extension as described herein, and may be half-life extended(for example because they comprise a Nanobody against human serumalbumin, such as (preferably) a Nanobody of the invention against humanserum albumin). Reference is made to Table X below. Generally, as TNF isa multivalent target, compounds of the invention comprising two or threeanti-TNF ISV's are preferred.

TABLE X Examples of compounds of the invention against TNF. Polypeptide/construct(¹) General formula ISV building blocks and linkers Monovalent[TNF] [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to 495Monovalent/ [TNF]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482to 495 C-terminal extension Monovalent/ [TNF]-L₁-[SA] [TNF] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[TNF] [SA] =(see legend below) extended L₁ = (see legend below) Monovalent/[TNF]-L₁-[SA]-X(n) [TNF] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[TNF]-X(n) [SA] = (see legend below) extended/C-L₁ = (see legend below) terminal X(n) = (see legend below) extensionBivalent(²) [TNF]-L₁-[TNF] At least one [TNF] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[TNF]-L₁-[TNF]-X(n) At least one = one of SEQ ID NO's: 464 to 477 and/orC-terminal 482 to 495 (⁴) extension(²) L₁ = (see legend below) X(n) =(see legend below) Bivalent/ [TNF]-L₁-[TNF]-L₂-[SA] At least one [TNF]present is = one of SEQ ID NO's: 464 to 477 half-life[TNF]-L₁-[SA]-L₂-[TNF] and/or 482 to 495 (⁴) extended(²)[SA]-L₁-[TNF]-L₂-[TNF] [SA] = (see legend below) L₁ = (see legend below)L₂ = (see legend below) Bivalent/ [TNF]-L₁-[TNF]-L₂-[SA]-X(n) At leastone [TNF] present is = one of SEQ ID NO's: 464 to 477 half-life[TNF]-L₁-[SA]-L₂-[TNF]-X(n) and/or 482 to 495 (⁴) extended/[SA]-L₁-[TNF]-L₂-[TNF]-X(n) [SA] = (see legend below) C-terminal L₁ =(see legend below) extension(²) L₂ = (see legend below) X(n) = (seelegend below) Bispecific (³) [TNF]-L₁-[Nb] At least one [TNF] present is= one of SEQ ID NO's: 464 to 477 [Nb]-L₁-[TNF] and/or 482 to 495 (⁴)[TNF]-L₁-[TNF]-L₂-[Nb] L₁ = (see legend below) [TNF]-L₁-[Nb]-L₂-[TNF] L₂= (see legend below) [Nb]-L₁-[TNF]-L₂-[TNF] [Nb] = (see legend below)Bispecific/ [TNF]-L₁-[Nb]-X(n) At least one[TNF] present is = one of SEQID NO's: 464 to 477 C-terminal [Nb]-L₁-[TNF]-X(n) and/or 482 to 495 (⁴)extension (3) [TNF]-L₁-[TNF]-L₂-[Nb]-X(n) L₁ = (see legend below)[TNF]-L₁-[Nb]-L₂-[TNF]-X(n) L₂ = (see legend below)[Nb]-L₁-[TNF]-L₂-[TNF]-X(n) X(n) = (see legend below) [Nb] = (see legendbelow) Bispecific/ [TNF]-L₁-[Nb]-L₂-[SA] At least one [TNF] present is =one of SEQ ID NO's: 464 to 477 half-life [TNF]-L₁-[SA]-L₂-[Nb] and/or482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[TNF] [SA] = (see legendbelow) [Nb]-L₁-[TNF]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[TNF]-L₂-[Nb] [Nb] = (see legend below) [SA]-L₁-[Nb]-L₂-[TNF]Bispecific/ [TNF]-L₁-[TNF]-L₂-[Nb]-L₃-[SA] At least one [TNF] present is= one of SEQ ID NO's: 464 to 477 half-life[Nb]-L₁-[TNF]-L₂-[TNF]-L₃-[SA] and/or 482 to 495 (⁴) extended (³)[SA]-L₁-[TNF]-L₂-[TNF]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[TNF]-L₂-[Nb]-L₃-[TNF] L₁, L₂, L₃(see legend below)[TNF]-L₁-[Nb]-L₂-[TNF]-L₃-[SA] [Nb] = (see legend below) Bispecific/[TNF]-L₁-[Nb]-L₂-[SA]-X(n) At least one [TNF] present is = one of SEQ IDNO's: 464 to 477 half-life [TNF]-L₁-[SA]-L₂-[Nb]-X(n) and/or 482 to 495(⁴) extended/ [Nb]-L₁-[SA]-L₂-[TNF]-X(n) [SA] = (see legend below)C-terminal [Nb]-L₁-[TNF]-L₂-[SA]-X(n) L₁, L₂, L₃(see legend below)extension (³) [SA]-L₁-[TNF]-L₂-[Nb]-X(n) X(n) = (see legend below)[SA]-L₁-[Nb]-L₂-[TNF]-X(n) [Nb] = (see legend below)[TNF]-L₁-[TNF]-L₂-[Nb]-L₃-[SA]-X(n) [Nb]-L₁-[TNF]-L₂-[TNF]-L₃-[SA]-X(n)[SA]-L₁-[TNF]-L₂-L₃-[Nb]-X(n) [SA]-L₁-[TNF]-L₂-[Nb]-L₃-[TNF]-X(n)[TNF]-L₁-[Nb]-L₂-[TNF]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD' s of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. NOTES: (¹)In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against TNF. These may further comprise a half-life extendingISV (such as an ISV against serum albumin). “Bivalent” generally refersto polypeptides/constructs comprising two ISV's against TNF (which maybe the same or different). These may again further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bispecific”generally refers to polypeptides/constructs comprising at least one(such as 1 or 2) ISV's against TNF and at least one (such as 1 or 2)other ISV against a therapeutic target. These may further comprise ahalf-life extending ISV (such as an ISV against serum albumin). In thepolypeptides/constructs described in this table, at least one of theISV's against TNF present is an ISV of the invention, and preferably allof the ISV's against TNF present in such polypeptide/construct are ISV'sof the invention. Also, when a half-life extending ISV and/or an ISVagainst another therapeutic target is present in suchpolypeptide/construct, each of these (and preferably all of these) mayalso be (and preferably are) ISV's of the invention (²)All “bivalent”constructs in this Table may also be biparatopic, meaning that theycomprise at least two (such as two) ISV's against TNF, which aredirected against different epitopes on TNF. (³) As will be clear to theskilled person, other/further bispecific constructs than those listedcan be made using the building blocks and linkers mentioned. (⁴)Preferably, each present is independently chosen from SEQ ID NO's: 464to 477 and/or 482 to 495. Also, the present may be the same ordifferent; in a biparatopic polypeptide/construct they will be directedagainst different epitopes on TNF. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 15: VH Domains (and in Particular Nanobodies) Against c-Met, andCompounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstc-Met.

Such a VH domain of the invention against c-Met will generally comprise:(i) suitable framework sequences that suitably comprise the amino acidresidues/mutations of the invention as described herein; as well as (ii)CDR sequences that allow the VH domain of the invention to specificallybind to c-Met. In addition, such a VH domain of the invention againstc-Met may also suitably have a C-terminal extension as described herein,in particular when said VH domain is monovalent or forms the C-terminalend of the compound of the invention in which said VH domain is present(again, as further described herein). Such VH domains of the inventionagainst c-Met may further be as further described herein, and may inparticular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against c-Met describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against c-Met, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against c-Met:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against c-Met may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against c-Met or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against c-Metcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 353(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 353; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 354 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 354; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 355 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 355.

More preferably, in a VH domain of the invention against c-Met accordingto this aspect: (i) CDR1 is SEQ ID NO: 353; (ii) CDR2 is SEQ ID NO: 354;and (iii) CDR3 is SEQ ID NO: 355.

In one specific aspect, a Nanobody of the invention against c-Met is avariant of the Nanobody of SEQ ID NO: 352 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 352),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstc-Met comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:371 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 371; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 372 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 372; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 373 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 373.

More preferably, in such a VH domain of the invention against c-Metaccording to this aspect: (i) CDR1 is SEQ ID NO: 371; (ii) CDR2 is SEQID NO: 372; and (iii) CDR3 is SEQ ID NO: 373.

In one specific aspect, a Nanobody of the invention against c-Met is avariant of the Nanobody of SEQ ID NO: 370 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 370),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against c-Met are listed in FIG. 18A as SEQ ID NO's: 356to 369 and in FIG. 18B as SEQ ID NO's: 374 to 387, respectively; andeach of these Nanobodies form a further aspect of the invention.

The invention also relates to a compound of the invention against c-Metthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 356 to 369 and/or 374 to387. Such compounds of the invention against c-Met may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table Ybelow.

As described in for example WO 2013/045707, one particularly preferredclass of Nanobody-based compounds against c-Met are biparatopiccompounds. Thus, in one aspect of the invention, a compound of theinvention against c-Met is a biparatopic construct that comprises oneISV that is either SEQ ID NO: 352 or (preferably) an ISV of theinvention that has been derived from SEQ ID NO: 352 (as described inthis Example 15) and one ISV that is either SEQ ID NO: 370 or(preferably) an ISV of the invention that has been derived from SEQ IDNO: 370 (as described in this Example 15), provided that at least one(and preferably both) of these ISV's are ISV's of the invention. Suchbiparatopic constructs may also be half-life extended (i.e. by means ofa serum albumin-binding ISV). Some specific examples of such biparatopicconstructs are given in SEQ ID NO: 676 to 693. Also, bispecificconstructs against c-Met may also comprise an ISV against VEGF or EGFR.Reference is again made to WO 2014/341309.

Some specifically preferred examples of compounds of the inventionagainst c-Met are given in FIGS. 28A and 28B SEQ ID NO's: 676 to 694;and each of these compounds form a further aspect of the invention.Thus, in another aspect, the invention relates to a polypeptide that isdirected against c-Met and that has an amino acid sequence that ischosen from the group consisting of SEQ ID NO's: 676 to 694. Moregenerally, compounds of the invention against c-Met may be as describedin WO 2013/045707, but comprising ISV's of the invention. They may alsobe used for the purposes described in WO 2013/045707.

TABLE Y Examples of compounds of the invention against c-Met.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent/ [c-Met] [c-Met] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [c-Met]-X(n) [c-Met] = one of SEQ ID NO's:464 to 477 and/or 482 to 495 C-terminal extension Monovalent/[c-Met]-L₁-[SA] [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 to495 half-life [SA]-L₁-[c-Met] [SA] = (see legend below) extended L₁=(see legend below) Monovalent/ [c-Met]-L₁-[SA]-X(n) [c-Met] = one of SEQID NO's: 464 to 477 and/or 482 to 495 half-life [SA]-L₁-[c-Met]-X(n)[SA] = (see legend below) extended/C- L₁ = (see legend below) terminalX(n) = (see legend below) extension Bivalent(²) [c-Met]-L₁-[c-Met] Atleast one [c-Met] present is = one of SEQ ID NO's: 464 to 477 and/or 482to 495 (⁴) L₁ = (see legend below) Bivalent/ [c-Met]-L₁-[c-Met]-X(n) Atleast one [c-Met] = one of SEQ ID NO's: 464 to 477 and/or 482 C-terminalto 495 (⁴) extension(²) L₁ = (see legend below) X(n) = (see legendbelow) Bivalent/ [c-Met]-L₁-[c-Met]-L₂-[SA] At least one [c-Met] presentis = one of SEQ ID NO's: 464 to 477 half-life [c-Met]-L₁-[SA]-L₂-[c-Met]and/or 482 to 495 (⁴) extended(²) [SA]-L₁-[c-Met]-L₂-[c-Met] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[c-Met]-L₁-[c-Met]-L₂-[SA]-X(n) At least one [c-Met] present is = one ofSEQ ID NO's: 464 to 477 half-life [c-Met]-L₁-[SA]-L₂-[c-Met]-X(n) and/or482 to 495 (⁴) extended/ [SA]-L₁-[c-Met]-L₂-[c-Met]-X(n) [SA] = (seelegend below) C-terminal L₁ = (see legend below) extension(²) L₂ = (seelegend below) X(n) = (see legend below) Bispecific (³) [c-Met]-L₁-[Nb]At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477[Nb]-L₁-[c-Met] and/or 482 to 495 (⁴) [c-Met]-L₁-[c-Met]-L₂-[Nb] L₁ =(see legend below) [c-Met]-L₁-[Nb]-L₂-[c-Met] L₂ = (see legend below)[Nb]-L₁-[c-Met]-L₂-[c-Met] [Nb] = (see legend below) Bispecific/[c-Met]-L₁-[Nb]-X(n) At least one [c-Met] present is = one of SEQ IDNO's: 464 to 477 C-terminal [Nb]-L₁-[c-Met]-X(n) and/or 482 to 495 (⁴)extension (3) [c-Met]-L₁-[c-Met]-L₂-[Nb]-X(n) L₁ = (see legend below)[c-Met]-L₁-[Nb]-L₂-[c-Met]-X(n) L₂ = (see legend below)[Nb]-L₁-[c-Met]-L₂-[c-Met]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [c-Met]-L₁-[Nb]-L₂-[SA] At least one [c-Met]present is = one of SEQ ID NO's: 464 to 477 half-life[c-Met]-L₁-[SA]-L₂-[Nb] and/or 482 to 495 (⁴) extended (³)[Nb]-L₁-[SA]-L₂-[c-Met] [SA] = (see legend below)[Nb]-L₁-[c-Met]-L₂-[SA] L₁, L₂, L₃(see legend below)[SA]-L₁-[c-Met]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[c-Met] Bispecific/ [c-Met]-L₁-[c-Met]-L₂-[Nb]-L₃-[SA]At least one [c-Met] present is = one of SEQ ID NO's: 464 to 477half-life [Nb]-L₁-[c-Met]-L₂-[c-Met]-L₃-[SA] and/or 482 to 495 (⁴)extended (³) [SA]-L₁-[c-Met]-L₂-[c-Met]-L₃-[Nb] [SA] = (see legendbelow) [SA]-L₁-[c-Met]-L₂-[Nb]-L₃-[c-Met] L₁, L₂, L₃(see legend below)[c-Met]-L₁-[Nb]-L₂-[c-Met]-L₃-[SA] [Nb] = (see legend below) Bispecific/[c-Met]-L₁-[Nb]-L₂-[SA]-X(n) At least one [c-Met] present is = one ofSEQ ID NO's: 464 to 477 half-life [c-Met]-L₁-[SA]-L₂-[Nb]-X(n) and/or482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[c-Met]-X(n) [SA] = (see legendbelow) C-terminal [Nb]-L₁-[c-Met]-L₂-[SA]-X(n) L₁, L₂, L₃(see legendbelow) extension (³) [SA]-L₁-[c-Met]-L₂-[Nb]-X(n) X(n) = (see legendbelow) [SA]-L₁-[Nb]-L₂-[c-Met]-X(n) [Nb] = (see legend below)[c-Met]-L₁-[c-Met]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[c-Met]-L₂-[c-Met]-L₃-[SA]-X(n)[SA]-L₁-[c-Met]-L₂-[c-Met]-L₃-[Nb]-X(n)[SA]-L₁-[c-Met]-L₂-[Nb]-L₃-[c-Met]-X(n)[c-Met]-L₁-[Nb]-L₂-[c-Met]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. Notes: (¹) In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against c-Met. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstc-Met (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against c-Met and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against c-Met present is an ISV of theinvention, and preferably all of the ISV's against c-Met present in suchpolypeptide/construct are ISV's of the invention. Also, when a half-lifeextending ISV and/or an ISV against another therapeutic target ispresent in such polypeptide/construct, each of these (and preferably allof these) may also be (and preferably are) ISV's of the invention (²):All “bivalent” constructs in this Table may also be biparatopic, meaningthat they comprise at least two (such as two) ISV's against c-Met, whichare directed against different epitopes on c-Met. (³): As will be clearto the skilled person, other/further bispecific constructs than thoselisted can be made using the building blocks and linkers mentioned. (⁴)Preferably, each [c-Met] present is independently chosen from SEQ IDNO's: 464 to 477 and/or 482 to 495. Also, the [c-Met] present may be thesame or different; in a biparatopic polypeptide/construct they will bedirected against different epitopes on c-Met. (⁵) Each of thepolypeptides/constructs mentioned to in this column by means ofreference to a SEQ ID forms an individual specific aspect of theinvention.

Example 16: VH Domains (and in Particular Nanobodies) Against RANK-L,and Compounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstRANK-L.

Such a VH domain of the invention against RANK-L will generallycomprise: (i) suitable framework sequences that suitably comprise theamino acid residues/mutations of the invention as described herein; aswell as (ii) CDR sequences that allow the VH domain of the invention tospecifically bind to RANK-L. In addition, such a VH domain of theinvention against RANK-L may also suitably have a C-terminal extensionas described herein, in particular when said VH domain is monovalent orforms the C-terminal end of the compound of the invention in which saidVH domain is present (again, as further described herein). Such VHdomains of the invention against RANK-L may further be as furtherdescribed herein, and may in particular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against RANK-L describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against RANK-L, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against RANK-L:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against RANK-L may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against RANK-L or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against RANK-Lcomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 389(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 389; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 390 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 390; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 391 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 391.

More preferably, in a VH domain of the invention against RANK-Laccording to this aspect: (i) CDR1 is SEQ ID NO: 389; (ii) CDR2 is SEQID NO: 390; and (iii) CDR3 is SEQ ID NO: 391.

In one specific aspect, a Nanobody of the invention against RANK-L is avariant of the Nanobody of SEQ ID NO: 388 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 388),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against RANK-L are listed in FIG. 19 as SEQ ID NO's: 392to 405; and each of these Nanobodies form a further aspect of theinvention.

The invention also relates to a compound of the invention against RANK-Lthat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 392 to 405. Such compoundsof the invention against RANK-L may again be as further describedherein, and thus for example may comprise suitable linkers, may comprisea C-terminal extension as described herein, and may be half-lifeextended (for example because they comprise a Nanobody against humanserum albumin, such as (preferably) a Nanobody of the invention againsthuman serum albumin). Reference is made to Table Z below.

Some specifically preferred examples of compounds of the inventionagainst RANK-L are given in FIG. 29 as SEQ ID NO's: 694 to 729; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst RANK-L and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 694 to 729.

More generally, compounds of the invention against RANK-L may be asdescribed in WO 2008/142164, but comprising ISV's of the invention. Theymay also be used for the purposes described in WO 2008/142164.

TABLE Z Examples of compounds of the invention against RANK-L.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [RANK-L] [RANK-L] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [RANK-L]-X(n) [RANK-L] = one of SEQ IDNO's: 464 to 477 and/or C-terminal 482 to 495 extension Monovalent/[RANK-L]-L₁-[SA] [RANK-L] = one of SEQ ID NO's: 464 to 477 and/orhalf-life [SA]-L₁-[RANK-L] 482 to 495 extended [SA] = (see legend below)L₁ = (see legend below) Monovalent/ [RANK-L]-L₁-[SA]-X(n) [RANK-L] = oneof SEQ ID NO's: 464 to 477 and/or half-life [SA]-L₁-[RANK-L]-X(n) 482 to495 extended/C- [SA] = (see legend below) terminal L₁ = (see legendbelow) extension X(n) = (see legend below) Bivalent(²)[RANK-L]-L₁-[RANK-L] At least one [RANK-L] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[RANK-L]-L₁-[RANK-L]-X(n) At least one [RANK-L] = one of SEQ ID NO's:464 to C-terminal 477 and/or 482 to 495 (⁴) extension(²) L₁ = (seelegend below) X(n) = (see legend below) Bivalent/[RANK-L]-L₁-[RANK-L]-L₂-[SA] At least one [RANK-L] present is = one ofSEQ ID NO's: half-life [RANK-L]-L₁-[SA]-L₂-[RANK-L] 464 to 477 and/or482 to 495 (⁴) extended(²) [SA]-L₁-[RANK-L]-L₂-[RANK-L] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[RANK-L]-L₁-[RANK-L]-L₂-[SA]-X(n) At least one [RANK-L] present is = oneof SEQ ID NO's: half-life [RANK-L]-L₁-[SA]-L₂-[RANK-L]-X(n) 464 to 477and/or 482 to 495 (⁴) extended/ [SA]-L₁-[RANK-L]-L₂-[RANK-L]-X(n) [SA] =(see legend below) C-terminal L₁ = (see legend below) extension(²) L₂ =(see legend below) X(n) = (see legend below) Bispecific (³)[RANK-L]-L₁-[Nb] At least one [RANK-L] present is = one of SEQ ID NO's:[Nb]-L₁-[RANK-L] 464 to 477 and/or 482 to 495 (⁴)[RANK-L]-L₁-[RANK-L]-L₂-[Nb] L₁ = (see legend below)[RANK-L]-L₁-[Nb]-L₂-[RANK-L] L₂ = (see legend below)[Nb]-L₁-[RANK-L]-L₂-[RANK-L] [Nb] = (see legend below) Bispecific/[RANK-L]-L₁-[Nb]-X(n) At least one [RANK-L] present is = one of SEQ IDNO's: C-terminal [Nb]-L₁-[RANK-L]-X(n) 464 to 477 and/or 482 to 495 (⁴)extension (3) [RANK-L]-L₁-[RANK-L]-L₂-[Nb]-X(n) L₁ = (see legend below)[RANK-L]-L₁-[Nb]-L₂-[RANK-L]-X(n) L₂ = (see legend below)[Nb]-L₁-[RANK-L]-L₂-[RANK-L]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [RANK-L]-L₁-[Nb]-L₂-[SA] At least one [RANK-L]present is = one of SEQ ID NO's: half-life [RANK-L]-L₁-[SA]-L₂-[Nb] 464to 477 and/or 482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[RANK-L] [SA]= (see legend below) [Nb]-L₁-[RANK-L]-L₂-[SA] L₁, L₂, L₃(see legendbelow) [SA]-L₁-[RANK-L]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[RANK-L] Bispecific/[RANK-L]-L₁-[RANK-L]-L₂-[Nb]-L₃-[SA] At least one [RANK-L] present is =one of SEQ ID NO's: half-life [Nb]-L₁-[RANK-L]-L₂-[RANK-L]-L₃-[SA] 464to 477 and/or 482 to 495 (⁴) extended (³)[SA]-L₁-[RANK-L]-L₂-[RANK-L]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[RANK-L]-L₂-[Nb]-L₃-[RANK-L] L₁, L₂, L₃(see legend below)[RANK-L]-L₁-[Nb]-L₂-[RANK-L]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [RANK-L]-L₁-[Nb]-L₂-[SA]-X(n) At least one [RANK-L] presentis = one of SEQ ID NO's: half-life [RANK-L]-L₁-[SA]-L₂-[Nb]-X(n) 464 to477 and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[RANK-L]-X(n) [SA] =(see legend below) C-terminal [Nb]-L₁-[RANK-L]-L₂-[SA]-X(n) L₁, L₂,L₃(see legend below) extension (³) [SA]-L₁-[RANK-L]-L₂-[Nb]-X(n) X(n) =(see legend below) [SA]-L₁-[Nb]-L₂-[RANK-L]-X(n) [Nb] = (see legendbelow) [RANK-L]-L₁-[RANK-L]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[RANK-L]-L₂-[RANK-L]-L₃-[SA]-X(n)[SA]-L₁-[RANK-L]-L₂-[RANK-L]-L₃-[Nb]-X(n)[SA]-L₁-[RANK-L]-L₂-[Nb]-L₃-[RANK-L]-X(n)[RANK-L]-L₁-[Nb]-L₂-[RANK-L]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. Notes: (¹) In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against RANK-L. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstRANK-L (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against RANK-L and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against RANK-L present is an ISV of theinvention, and preferably all of the ISV's against RANK-L present insuch polypeptide/construct are ISV's of the invention. Also, when ahalf-life extending ISV and/or an ISV against another therapeutic targetis present in such polypeptide/construct, each of these (and preferablyall of these) may also be (and preferably are) ISV's of the invention(²): All “bivalent” constructs in this Table may also be biparatopic,meaning that they comprise at least two (such as two) ISV's againstRANK-L, which are directed against different epitopes on RANK-L. (³): Aswill be clear to the skilled person, other/further bispecific constructsthan those listed can be made using the building blocks and linkersmentioned. (⁴) Preferably, each [RANK-L] present is independently chosenfrom SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [RANK-L]present may be the same or different; in a biparatopicpolypeptide/construct they will be directed against different epitopeson RANK-L. (⁵) Each of the polypeptides/constructs mentioned to in thiscolumn by means of reference to a SEQ ID forms an individual specificaspect of the invention.

Example 17: VH Domains (and in Particular Nanobodies) Against CXCR-7,and Compounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstCXCR-7.

Such a VH domain of the invention against CXCR-7 will generallycomprise: (i) suitable framework sequences that suitably comprise theamino acid residues/mutations of the invention as described herein; aswell as (ii) CDR sequences that allow the VH domain of the invention tospecifically bind to CXCR-7. In addition, such a VH domain of theinvention against CXCR-7 may also suitably have a C-terminal extensionas described herein, in particular when said VH domain is monovalent orforms the C-terminal end of the compound of the invention in which saidVH domain is present (again, as further described herein). Such VHdomains of the invention against CXCR-7 may further be as furtherdescribed herein, and may in particular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against CXCR-7 describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against CXCR-7, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against CXCR-7:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against CXCR-7 may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against CXCR-7 or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against CXCR-7comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 407(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 407; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 408 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 408; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 409 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 409.

More preferably, in a VH domain of the invention against CXCR-7according to this aspect: (i) CDR1 is SEQ ID NO: 407; (ii) CDR2 is SEQID NO: 408; and (iii) CDR3 is SEQ ID NO: 409.

In one specific aspect, a Nanobody of the invention against CXCR-7 is avariant of the Nanobody of SEQ ID NO: 406 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 406),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstCXCR-7 comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:425 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 425; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 426 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 426; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 427 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 427.

More preferably, in a VH domain of the invention against CXCR-7according to this aspect: (i) CDR1 is SEQ ID NO: 425; (ii) CDR2 is SEQID NO: 426; and (iii) CDR3 is SEQ ID NO: 427.

In one specific aspect, a Nanobody of the invention against CXCR-7 is avariant of the Nanobody of SEQ ID NO: 424 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 424),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against CXCR-7 are listed in FIG. 20A as SEQ ID NO's: 410to 423 and in FIG. 20B as SEQ ID NO's: 428 to 441; and each of theseNanobodies form a further aspect of the invention.

The invention also relates to a compound of the invention against CXCR-7that comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 410 to 423 and/or 428 to441. Such compounds of the invention against CXCR-7 may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table AAbelow.

As described in for example WO2012/130874, one particularly preferredclass of Nanobody-based compounds against CXCR7 are biparatopiccompounds. Thus, in one aspect of the invention, a compound of theinvention against CXCR-7 is a biparatopic construct that comprises oneISV that is either SEQ ID NO: 406 or (preferably) an ISV of theinvention that has been derived from SEQ ID NO: 406 (as described inthis Example 17) and one ISV that is either SEQ ID NO: 424 or(preferably) an ISV of the invention that has been derived from SEQ IDNO: 424 (as described in this Example 17), provided that at least one(and preferably both) of these ISV's are ISV's of the invention. Suchbiparatopic constructs may also be half-life extended (i.e. by means ofa serum albumin-binding ISV).

More generally, compounds of the invention against CXCR-7 may be asdescribed in WO2012/130874, but comprising ISV's of the invention. Theymay also be used for the purposes described in WO2012/130874.

TABLE AA Examples of compounds of the invention against CXCR-7.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [CXCR-7] [CXCR-7] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [CXCR-7]-X(n) [CXCR-7] = one of SEQ IDNO's: 464 to 477 and/or C-terminal 482 to 495 extension Monovalent/[CXCR-7]-L₁-[SA] [CXCR-7] = one of SEQ ID NO's: 464 to 477 and/orhalf-life [SA]-L₁-[CXCR-7] 482 to 495 extended [SA] = (see legend below)L₁ = (see legend below) Monovalent/ [CXCR-7]-L₁-[SA]-X(n) [CXCR-7] = oneof SEQ ID NO's: 464 to 477 and/or half-life [SA]-L₁-[CXCR-7]-X(n) 482 to495 extended/C- [SA] = (see legend below) terminal L₁ = (see legendbelow) extension X(n) = (see legend below) Bivalent(²)[CXCR-7]-L₁-[CXCR-7] At least one [CXCR-7] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[CXCR-7]-L₁-[CXCR-7]-X(n) At least one [CXCR-7] = one of SEQ ID NO's:464 to C-terminal 477 and/or 482 to 495 (⁴) extension(²) L₁ = (seelegend below) X(n) = (see legend below) Bivalent/[CXCR-7]-L₁-[CXCR-7]-L₂-[SA] At least one [CXCR-7] present is = one ofSEQ ID NO's: half-life [CXCR-7]-L₁-[SA]-L₂-[CXCR-7] 464 to 477 and/or482 to 495 (⁴) extended(²) [SA]-L₁-[CXCR-7]-L₂-[CXCR-7] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[CXCR-7]-L₁-[CXCR-7]-L₂-[SA]-X(n) At least one [CXCR-7] present is = oneof SEQ ID NO's: half-life [CXCR-7]-L₁-[SA]-L₂-[CXCR-7]-X(n) 464 to 477and/or 482 to 495 (⁴) extended/ [SA]-L₁-[CXCR-7]-L₂-[CXCR-7]-X(n) [SA] =(see legend below) C-terminal L₁ = (see legend below) extension(²) L₂ =(see legend below) X(n) = (see legend below) Bispecific (³)[CXCR-7]-L₁-[Nb] At least one [CXCR-7] present is = one of SEQ ID NO's:[Nb]-L₁-[CXCR-7] 464 to 477 and/or 482 to 495 (⁴)[CXCR-7]-L₁-[CXCR-7]-L₂-[Nb] L₁ = (see legend below)[CXCR-7]-L₁-[Nb]-L₂-[CXCR-7] L₂ = (see legend below)[Nb]-L₁-[CXCR-7]-L₂-[CXCR-7] [Nb] = (see legend below) Bispecific/[CXCR-7]-L₁-[Nb]-X(n) At least one [CXCR-7] present is = one of SEQ IDNO's: C-terminal [Nb]-L₁-[CXCR-7]-X(n) 464 to 477 and/or 482 to 495 (⁴)extension (3) [CXCR-7]-L₁-[CXCR-7]-L₂-[Nb]-X(n) L₁ = (see legend below)[CXCR-7]-L₁-[Nb]-L₂-[CXCR-7]-X(n) L₂ = (see legend below)[Nb]-L₁-[CXCR-7]-L₂-[CXCR-7]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [CXCR-7]-L₁-[Nb]-L₂-[SA] At least one [CXCR-7]present is = one of SEQ ID NO's: half-life [CXCR-7]-L₁-[SA]-L₂-[Nb] 464to 477 and/or 482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[CXCR-7] [SA]= (see legend below) [Nb]-L₁-[CXCR-7]-L₂-[SA] L₁, L₂, L₃(see legendbelow) [SA]-L₁-[CXCR-7]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[CXCR-7] Bispecific/[CXCR-7]-L₁-[CXCR-7]-L₂-[Nb]-L₃-[SA] At least one [CXCR-7] present is =one of SEQ ID NO's: half-life [Nb]-L₁-[CXCR-7]-L₂-[CXCR-7]-L₃-[SA] 464to 477 and/or 482 to 495 (⁴) extended (³)[SA]-L₁-[CXCR-7]-L₂-[CXCR-7]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[CXCR-7]-L₂-[Nb]-L₃-[CXCR-7] L₁, L₂, L₃(see legend below)[CXCR-7]-L₁-[Nb]-L₂-[CXCR-7]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [CXCR-7]-L₁-[Nb]-L₂-[SA]-X(n) At least one [CXCR-7] presentis = one of SEQ ID NO's: half-life [CXCR-7]-L₁-[SA]-L₂-[Nb]-X(n) 464 to477 and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[CXCR-7]-X(n) [SA] =(see legend below) C-terminal [Nb]-L₁-[CXCR-7]-L₂-[SA]-X(n) L₁, L₂,L₃(see legend below) extension (³) [SA]-L₁-[CXCR-7]-L₂-[Nb]-X(n) X(n) =(see legend below) [SA]-L₁-[Nb]-L₂-[CXCR-7]-X(n) [Nb] = (see legendbelow) [CXCR-7]-L₁-[CXCR-7]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[CXCR-7]-L₂-[CXCR-7]-L₃-[SA]-X(n)[SA]-L₁-[CXCR-7]-L₂-[CXCR-7]-L₃-[Nb]-X(n)[SA]-L₁-[CXCR-7]-L₂-[Nb]-L₃-[CXCR-7]-X(n)[CXCR-7]-L₁-[Nb]-L₂-[CXCR-7]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. Notes: (¹) In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against CXCR-7. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstCXCR-7 (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against CXCR-7 and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against CXCR-7 present is an ISV of theinvention, and preferably all of the ISV's against CXCR-7 present insuch polypeptide/construct are ISV's of the invention. Also, when ahalf-life extending ISV and/or an ISV against another therapeutic targetis present in such polypeptide/construct, each of these (and preferablyall of these) may also be (and preferably are) ISV's of the invention(²): All “bivalent” constructs in this Table may also be biparatopic,meaning that they comprise at least two (such as two) ISV's againstCXCR-7, which are directed against different epitopes on CXCR-7. (³): Aswill be clear to the skilled person, other/further bispecific constructsthan those listed can be made using the building blocks and linkersmentioned. (⁴) Preferably, each [CXCR-7] present is independently chosenfrom SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [CXCR-7]present may be the same or different; in a biparatopicpolypeptide/construct they will be directed against different epitopeson CXCR-7. (⁵) Each of the polypeptides/constructs mentioned to in thiscolumn by means of reference to a SEQ ID forms an individual specificaspect of the invention.

Example 18: VH Domains (and in Particular Nanobodies) Against A-Beta,and Compounds of the Invention Comprising the Same

In one specific aspect, the VH domains of the invention (and inparticular ISVDs of the invention and more in particular Nanobodies ofthe invention) and compounds of the invention may be directed againstA-beta.

Such a VH domain of the invention against A-beta will generallycomprise: (i) suitable framework sequences that suitably comprise theamino acid residues/mutations of the invention as described herein; aswell as (ii) CDR sequences that allow the VH domain of the invention tospecifically bind to A-beta. In addition, such a VH domain of theinvention against A-beta may also suitably have a C-terminal extensionas described herein, in particular when said VH domain is monovalent orforms the C-terminal end of the compound of the invention in which saidVH domain is present (again, as further described herein). Such VHdomains of the invention against A-beta may further be as furtherdescribed herein, and may in particular be ISVD's.

Again, as with other aspects and embodiments of the invention describedherein, when a specific ISVD (such as the ISVD against A-beta describedin this Example) or compound comprising the same is said to be“according to the invention” or “as further described herein”, thepreferred aspects/embodiments and preferences that are generallydescribed herein for the ISVD's or compounds of the invention alsospecifically apply to said specific ISVD or compound, respectively,unless explicitly indicated otherwise or unless the specific technicalcontext requires otherwise.

Thus, in a particular aspect, the present invention relates to a VHdomain (and in particular an ISVD) that is directed against A-beta, inwhich (i) position 112 is K or Q; or (ii) position 110 is K or Q andposition 11 is V; or (iii) position 89 is T; or (iv) position 89 is Land position 110 is K or Q; or (v) position 11 is V and position 89 isL; or any suitable combination of (i) to (v). In particular, in such VHdomains against A-beta:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v).

The VH domains of the invention against A-beta may further be asdescribed herein and may again in particular be an ISVD (and more inparticular a Nanobody) against A-beta or a protein, polypeptide or othercompound or construct that comprises as least one such ISVD. Such aprotein, polypeptide or other compound or construct may also be asfurther described herein, and may for example have an increasedhalf-life (i.e. as described herein, e.g. a half-life—expressed as t½beta—in human subjects of in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days), and for this purpose may for example comprise aserum-albumin binding Nanobody, which may also be a serum-albuminbinding Nanobody of the invention (again, as described herein).

Also, such an ISVD may suitably have a C-terminal extension (as furtherdescribed herein and in WO 12/175741), in particular when said ISVDforms the C-terminal end of a protein, polypeptide or other compound orconstruct comprising the same (again, as further described herein).

In one preferred aspect, the VH domains of the invention against A-betacomprise (i) a CDR1 sequence that is the sequence of SEQ ID NO: 461(which is preferred) or that is an amino acid sequence that has only oneamino acid difference with the sequence of SEQ ID NO: 461; (ii) a CDR2sequence that is the sequence of SEQ ID NO: 462 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 462; and (iii) a CDR3sequence that is the sequence of SEQ ID NO: 463 (which is preferred) orthat is an amino acid sequence that has only one or two amino aciddifferences with the sequence of SEQ ID NO: 463.

More preferably, in a VH domain of the invention against A-betaaccording to this aspect: (i) CDR1 is SEQ ID NO: 461; (ii) CDR2 is SEQID NO: 462; and (iii) CDR3 is SEQ ID NO: 463.

In one specific aspect, a Nanobody of the invention against A-beta is avariant of the Nanobody of SEQ ID NO: 460 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 460),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). The CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

In another preferred aspect, the VH domains of the invention againstA-beta comprise (i) a CDR1 sequence that is the sequence of SEQ ID NO:479 (which is preferred) or that is an amino acid sequence that has onlyone amino acid difference with the sequence of SEQ ID NO: 479; (ii) aCDR2 sequence that is the sequence of SEQ ID NO: 480 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 480; and (iii) aCDR3 sequence that is the sequence of SEQ ID NO: 481 (which ispreferred) or that is an amino acid sequence that has only one or twoamino acid differences with the sequence of SEQ ID NO: 481.

More preferably, in a VH domain of the invention against A-betaaccording to this aspect: (i) CDR1 is SEQ ID NO: 479; (ii) CDR2 is SEQID NO: 480; and (iii) CDR3 is SEQ ID NO: 481.

In one specific aspect, a Nanobody of the invention against A-beta is avariant of the Nanobody of SEQ ID NO: 478 (with at least 90% sequenceidentity, such as at least 95% sequence identity, with SEQ ID NO: 478),in which:

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q;        such that either (i) position 112 is K or Q; or (ii) position        110 is K or Q and position 11 is V; or (iii) position 89 is T;        or (iv) position 89 is L and position 110 is K or Q; or (v)        position 11 is V and position 89 is L; or any suitable        combination of (i) to (v). Again, the CDR's of such an ISV are        preferably as defined in the preceding two paragraphs.

Some specifically preferred, but non-limiting examples of Nanobodies ofthe invention against A-beta are listed in FIG. 21A as SEQ ID NO's: 464to 477 and in FIG. 21B as SEQ ID NO's: 482 to 495; and each of theseNanobodies form a further aspect of the invention.

The invention also relates to a compound of the invention against A-betathat comprises at least one (such as one, two or three) of theNanobodies of the invention of SEQ ID NO's: 464 to 477 and/or 482 to495. Such compounds of the invention against A-beta may again be asfurther described herein, and thus for example may comprise suitablelinkers, may comprise a C-terminal extension as described herein, andmay be half-life extended (for example because they comprise a Nanobodyagainst human serum albumin, such as (preferably) a Nanobody of theinvention against human serum albumin). Reference is made to Table BBbelow.

As described in for example WO 2006/040153 and in particular asdescribed in EP2542579, one particularly preferred class ofNanobody-based compounds against A-beta are biparatopic compounds. Thus,in one aspect of the invention, a compound of the invention againstA-beta is a biparatopic construct that comprises one ISV that is eitherSEQ ID NO: 460 or (preferably) an ISV of the invention that has beenderived from SEQ ID NO: 460 (as described in this Example 17) and oneISV that is either SEQ ID NO: 478 or (preferably) an ISV of theinvention that has been derived from SEQ ID NO: 478 (as described inthis Example 17), provided that at least one (and preferably both) ofthese ISV's are ISV's of the invention. Such biparatopic constructs mayalso be half-life extended (i.e. by means of a serum albumin-bindingISV). Some specific examples of such biparatopic constructs are given inSEQ ID NO: 730 to 766.

Some specifically preferred examples of compounds of the inventionagainst A-beta are given in FIG. 30 as SEQ ID NO's: 730 to 766; and eachof these compounds form a further aspect of the invention. Thus, inanother aspect, the invention relates to a polypeptide that is directedagainst A-beta and that has an amino acid sequence that is chosen fromthe group consisting of SEQ ID NO's: 730 to 766. More generally,compounds of the invention against A-beta may be as described in WO2006/040153 and in particular as described in EP2542579, but comprisingISV's of the invention. They may also be used for the purposes describedin WO 2006/040153 and in particular EP2542579.

TABLE BB Examples of compounds of the invention against A-beta.Polypeptide/ construct(¹) General formula ISV building blocks andlinkers Monovalent [A-beta] [A-beta] = one of SEQ ID NO's: 464 to 477and/or 482 to 495 Monovalent/ [A-beta]-X(n) [A-beta] = one of SEQ IDNO's: 464 to 477 and/or C-terminal 482 to 495 extension Monovalent/[A-beta]-L₁-[SA] [A-beta] = one of SEQ ID NO's: 464 to 477 and/orhalf-life [SA]-L₁-[A-beta] 482 to 495 extended [SA] = (see legend below)L₁ = (see legend below) Monovalent/ [A-beta]-L₁-[SA]-X(n) [A-beta] = oneof SEQ ID NO's: 464 to 477 and/or half-life [SA]-L₁-[A-beta]-X(n) 482 to495 extended/C- [SA] = (see legend below) terminal L₁ = (see legendbelow) extension X(n) = (see legend below) Bivalent(²)[A-beta]-L₁-[A-beta] At least one [A-beta] present is = one of SEQ IDNO's: 464 to 477 and/or 482 to 495 (⁴) L₁ = (see legend below) Bivalent/[A-beta]-L₁-[A-beta]-X(n) At least one [A-beta] = one of SEQ ID NO's:464 to C-terminal 477 and/or 482 to 495 (⁴) extension(²) L₁ = (seelegend below) X(n) = (see legend below) Bivalent/[A-beta]-L₁-[A-beta]-L₂-[SA] At least one [A-beta] present is = one ofSEQ ID NO's: half-life [A-beta]-L₁-[SA]-L₂-[A-beta] 464 to 477 and/or482 to 495 (⁴) extended(²) [SA]-L₁-[A-beta]-L₂-[A-beta] [SA] = (seelegend below) L₁ = (see legend below) L₂ = (see legend below) Bivalent/[A-beta]-L₁-[A-beta]-L₂-[SA]-X(n) At least one [A-beta] present is = oneof SEQ ID NO's: half-life [A-beta]-L₁-[SA]-L₂-[A-beta]-X(n) 464 to 477and/or 482 to 495 (⁴) extended/ [SA]-L₁-[A-beta]-L₂-[A-beta]-X(n) [SA] =(see legend below) C-terminal L₁ = (see legend below) extension(²) L₂ =(see legend below) X(n) = (see legend below) Bispecific (³)[A-beta]-L₁-[Nb] At least one [A-beta] present is = one of SEQ ID NO's:[Nb]-L₁-[A-beta] 464 to 477 and/or 482 to 495 (⁴)[A-beta]-L₁-[A-beta]-L₂-[Nb] L₁ = (see legend below)[A-beta]-L₁-[Nb]-L₂-[A-beta] L₂ = (see legend below)[Nb]-L₁-[A-beta]-L₂-[A-beta] [Nb] = (see legend below) Bispecific/[A-beta]-L₁-[Nb]-X(n) At least one [A-beta] present is = one of SEQ IDNO's: C-terminal [Nb]-L₁-[A-beta]-X(n) 464 to 477 and/or 482 to 495 (⁴)extension (3) [A-beta]-L₁-[A-beta]-L₂-[Nb]-X(n) L₁ = (see legend below)[A-beta]-L₁-[Nb]-L₂-[A-beta]-X(n) L₂ = (see legend below)[Nb]-L₁-[A-beta]-L₂-[A-beta]-X(n) X(n) = (see legend below) [Nb] = (seelegend below) Bispecific/ [A-beta]-L₁-[Nb]-L₂-[SA] At least one [A-beta]present is = one of SEQ ID NO's: half-life [A-beta]-L₁-[SA]-L₂-[Nb] 464to 477 and/or 482 to 495 (⁴) extended (³) [Nb]-L₁-[SA]-L₂-[A-beta] [SA]= (see legend below) [Nb]-L₁-[A-beta]-L₂-[SA] L₁, L₂, L₃(see legendbelow) [SA]-L₁-[A-beta]-L₂-[Nb] [Nb] = (see legend below)[SA]-L₁-[Nb]-L₂-[A-beta] Bispecific/[A-beta]-L₁-[A-beta]-L₂-[Nb]-L₃-[SA] At least one [A-beta] present is =one of SEQ ID NO's: half-life [Nb]-L₁-[A-beta]-L₂-[A-beta]-L₃-[SA] 464to 477 and/or 482 to 495 (⁴) extended (³)[SA]-L₁-[A-beta]-L₂-[A-beta]-L₃-[Nb] [SA] = (see legend below)[SA]-L₁-[A-beta]-L₂-[Nb]-L₃-[A-beta] L₁, L₂, L₃(see legend below)[A-beta]-L₁-[Nb]-L₂-[A-beta]-L₃-[SA] [Nb] = (see legend below)Bispecific/ [A-beta]-L₁-[Nb]-L₂-[SA]-X(n) At least one [A-beta] presentis = one of SEQ ID NO's: half-life [A-beta]-L₁-[SA]-L₂-[Nb]-X(n) 464 to477 and/or 482 to 495 (⁴) extended/ [Nb]-L₁-[SA]-L₂-[A-beta]-X(n) [SA] =(see legend below) C-terminal [Nb]-L₁-[A-beta]-L₂-[SA]-X(n) L₁, L₂,L₃(see legend below) extension (³) [SA]-L₁-[A-beta]-L₂-[Nb]-X(n) X(n) =(see legend below) [SA]-L₁-[Nb]-L₂-[12345]-X(n) [Nb] = (see legendbelow) [A-beta]-L₁-[A-beta]-L₂-[Nb]-L₃-[SA]-X(n)[Nb]-L₁-[A-beta]-L₂-[A-beta]-L₃-[SA]-X(n)[SA]-L₁-[A-beta]-L₂-[A-beta]-L₃-[Nb]-X(n)[SA]-L₁-[A-beta]-L₂-[Nb]-L₃-[A-beta]-X(n)[A-beta]-L₁-[Nb]-L₂-[A-beta]-L₃-[SA]-X(n) Legend: [SA] is an ISV against(human) serum albumin, preferably an ISV of the invention against(human) serum albumin, more preferably one of SEQ ID NO's: 46 or 61 oreven more preferably one of the ISVD's of the invention of SEQ ID NO's:47, 54, 62, 69, 78, 86, 109, 116, 123, 130 or 496 to 513. Each of L₁, L₂and L₃ is (independently) a suitable linker. Each of L₁, L₂ and L₃ may(independently) be present or not. Non-limiting examples of suitablelinkers are the gly-ser linkers referred to herein, such as the 9GS,30GS or 35GS linker. X(n) = a C-terminal extension as a C-terminalextension as described in herein and/or in WO 12/175741 [Nb] is an ISVagainst another therapeutic target. Notes: (¹) In this Table:“Monovalent” generally refers to polypeptides/constructs comprising asingle ISV against A-beta. These may further comprise a half-lifeextending ISV (such as an ISV against serum albumin). “Bivalent”generally refers to polypeptides/constructs comprising two ISV's againstA-beta (which may be the same or different). These may again furthercomprise a half-life extending ISV (such as an ISV against serumalbumin). “Bispecific” generally refers to polypeptides/constructscomprising at least one (such as 1 or 2) ISV's against A-beta and atleast one (such as 1 or 2) other ISV against a therapeutic target. Thesemay further comprise a half-life extending ISV (such as an ISV againstserum albumin). In the polypeptides/constructs described in this table,at least one of the ISV's against A-beta present is an ISV of theinvention, and preferably all of the ISV's against A-beta present insuch polypeptide/construct are ISV's of the invention. Also, when ahalf-life extending ISV and/or an ISV against another therapeutic targetis present in such polypeptide/construct, each of these (and preferablyall of these) may also be (and preferably are) ISV's of the invention(²): All “bivalent” constructs in this Table may also be biparatopic,meaning that they comprise at least two (such as two) ISV's againstA-beta, which are directed against different epitopes on A-beta. (³): Aswill be clear to the skilled person, other/further bispecific constructsthan those listed can be made using the building blocks and linkersmentioned. (⁴) Preferably, each [A-beta] present is independently chosenfrom SEQ ID NO's: 464 to 477 and/or 482 to 495. Also, the [A-beta]present may be the same or different; in a biparatopicpolypeptide/construct they will be directed against different epitopeson A-beta. (⁵) Each of the polypeptides/constructs mentioned to in thiscolumn by means of reference to a SEQ ID forms an individual specificaspect of the invention.

Example 19: Testing of Anti-A-Beta Constructs for Binding byPre-Existing Antibodies

Three half-life extended anti-A-beta constructs (with the generalformula [A-beta-1]-9GS-ALB8-9GS-[A-beta-2]-A) were tested and comparedfor binding by pre-existing antibodies, using the general protocoldescribed herein. The two constructs according to the invention had theindicated mutations of the invention in all three building blocks (i.e.in the two anti-A-beta Nanobodies and in the serum albumin-bindingNanobody). The reference construct (SEQ ID NO:766) had no mutations ofthe invention in any of the building blocks. All constructs tested has aC-terminal alanine. The results are given in Table CC-1 and FIG. 31A

TABLE CC-1 testing of anti-A-beta constructs for binding by pre-existingantibodies Nanobodies Ref. Binding Binding Binding tested on 92 no. inLevel at Level at Level at samples FIG. 125 seconds 125 seconds 125seconds (healthy subjects) 30A <10 RU <20 RU >20 RU SEQ ID NO: 766 (1) 01 91 (reference) SEQ ID NO: 733 (2) 11 41 51 (invention: L11V + V89L)SEQ ID NO: 749 (3) 16 56 36 (invention: L11V + V89L + T110K)

The anti-A-beta Nanobodies that were present at the C-terminal end ofthe constructs were also tested separately as monovalent constructs(with a C-terminal alanine). The results are given in Table CC-2 andFIG. 31A.

TABLE CC-2 testing of monovalent anti-A-beta Nanobodies for binding bypre-existing antibodies Nanobodies Ref. Binding Binding Binding testedon 145 no. in Level at Level at Level at samples FIG. 125 seconds 125seconds 125 seconds (healthy subjects) 30A <10 RU <20 RU >20 RU SEQ IDNO: 478 (1) 67 87 58 (reference) (*) SEQ ID NO: 489 (2) 74 103 42(invention: L11V + V89L) (*) SEQ ID NO: 490 (3) 116 138 7 (invention:L11V + V89L + T110K) (*) (*) all three monovalent Nanobodies tested hada C-terminal alanine added

Example 20: Overview of Serum Albumin Binders of the Invention

Tables DD and EE below give some preferred, but non-limiting examples ofserum albumin binding Nanobodies of the invention, based on Alb-8 (TableDD) and Alb-23 (Table EE), respectively.

As mentioned, the invention also relates to a polypeptide, protein,compound or construct (and in particular a compound of the invention)that comprises one of the serum albumin-binding Nanobodies listed inTable DD or EE below. Such a polypeptide, protein, compound or construct(and in particular a compound of the invention) may further comprise atleast one (such as one, two or three) binding domain or binding unit(such as an ISVD, and in particular an ISV of the invention) that isdirected against at least one therapeutic target. Such a polypeptide,protein, compound or construct may again suitably be monospecific,bispecific or trispecific with respect to the therapeutic target(s), andmay be bivalent, trivalent, tetravalent or of higher valency. It willusually also contain suitable linkers, and may comprise a C-terminalextension as described herein.

In particular, such a polypeptide, protein, compound or construct may bea compound of the invention (as described herein) and/or may be asfurther described herein for the compounds of the invention (includingpreferred embodiments for compounds of the invention. Thus compoundscomprising ISVD's and in particular Nanobodies are particularlypreferred). Accordingly compounds of the invention comprising one of theserum albumin binding Nanobodies listed in Table DD or EE form furtheraspects of the invention.

TABLE DD Alb-8 (reference) and Alb-8 variants according to the invention SEQ IDNO: Variant Sequence 46 Alb-8EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL (WOEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT 06/122787)AVYYCTIGGSLSRSSQGTLVTVSS 496 89L + 110KEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT VALYYCTIGGSLSRSSQGTLVKVSS497 89L + 110Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVQVSS498 110K EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVKVSS499 110Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVQVSS47 112K EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVKS54 112Q EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVQS78 89T EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT ATYYCTIGGSLSRSSQGTLVTVSS109 11V + 89L EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TALYYCTIGGSLSRSSQGTLVTVSS123 11V + 89L + EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL 110KEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TALYYCTIGGSLSRSSQGTLVKVSS500 11V + 89L + EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGL 110QEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TALYYCTIGGSLSRSSQGTLVQVSS501 11V + 110K EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED VTAVYYCTIGGSLSRSSQGTLVKVSS502 11V + 110Q EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVQVSS503 11V + 112K EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVTVKS504 11V + 112Q EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVTVQS

TABLE EEAlb-23 (reference) and Alb-23 variants according to the invention SEQ IDNO: Variant Sequence 61 Alb-23EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGP (WOEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT 12/175400)AVYYCTIGGSLSRSSQGTLVTVSS 505 89L + 110KEVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVKVSS506 89L + 110Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVQVSS507 110K EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVKVSS508 110Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVQVSS62 112K EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVKS69 112Q EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVQS86 89T EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ATYYCTIGGSLSRSSQGTLVTVSS116 11V + 89L EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVTVSS130 11V + 89L + EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGP 110KEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVKVSS509 11V + 89L + EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGP 110QEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT ALYYCTIGGSLSRSSQGTLVQVSS510 11V + 110K EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVKVSS511 11V + 110Q EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVQVSS512 11V + 112K EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVKS513 11V + 112Q EVQLLESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVQS

The invention claimed is:
 1. A method of modifying an immunoglobulinsingle variable domain (VH or VHH) to have reduced binding bypre-existing antibodies found in human blood or serum, the methodcomprising mutating the amino acid sequence of the immunoglobulin singlevariable domain to generate a modified immunoglobulin single variabledomain comprising the amino acid V at position 11 and the amino acid Lat position 89, wherein the positions are numbered according to Kabatnumbering; wherein, prior to mutation, the immunoglobulin singlevariable domain comprises: (i) an amino acid other than V at position 11and an amino acid other than L at position 89; (ii) an amino acid V atposition 11 and an amino acid other than L at position 89; or (iii) anamino acid other than V at position 11 and an amino acid L at position89.
 2. The method of claim 1, wherein the modified immunoglobulin singlevariable domain comprises an amino acid K or Q at position 110 accordingto Kabat numbering.
 3. The method of claim 2, further comprisingextending the C-terminal end of said immunoglobulin single variabledomain by 1 to 5 amino acid residues.
 4. The method of claim 2, whereinthe modified immunoglobulin single variable domain comprises an aminoacid K or Q at position 112 according to Kabat numbering.
 5. The methodof claim 4, further comprising extending the C-terminal end of saidimmunoglobulin single variable domain by 1 to 5 amino acid residues. 6.The method of claim 1, wherein the modified immunoglobulin singlevariable domain comprises an amino acid K or Q at position 112 accordingto Kabat numbering.
 7. The method of claim 6, further comprisingextending the C-terminal end of said immunoglobulin single variabledomain by 1 to 5 amino acid residues.
 8. The method of claim 1, whereinthe modified immunoglobulin single variable domain comprises an aminoacid A or P at position 14 according to Kabat numbering.
 9. The methodof claim 1, wherein the modified immunoglobulin single variable domaincomprises an amino acid A or P at position 41 according to Kabatnumbering.
 10. The method of claim 1, wherein the modifiedimmunoglobulin single variable domain comprises an amino acid Q or L atposition 108 according to Kabat numbering.
 11. The method of claim 1,wherein the immunoglobulin single variable domain is comprised in aprotein, polypeptide or other construct.
 12. The method of claim 1,further comprising extending the C-terminal end of said immunoglobulinsingle variable domain by 1 to 5 amino acid residues.
 13. A method ofmodifying an immunoglobulin single variable domain (VH or VHH) having anamino acid other than K or Q at position 112 to have reduced binding bypre-existing antibodies found in human blood or serum, the methodcomprising mutating the amino acid at position 112 to K or Q, whereinthe positions are numbered according to Kabat numbering, optionallywherein the modified immunoglobulin single variable domain comprises anamino acid Q or L at position 108 according to Kabat numbering.
 14. Themethod of claim 13, further comprising extending the C-terminal end ofsaid immunoglobulin single variable domain by 1 to 5 amino acidresidues.
 15. The method of claim 13, wherein the modifiedimmunoglobulin single variable domain comprises an amino acid K or Q atposition 110 according to Kabat numbering.
 16. The method of claim 15,further comprising extending the C-terminal end of said immunoglobulinsingle variable domain by 1 to 5 amino acid residues.
 17. The method ofclaim 13, wherein the modified immunoglobulin single variable domaincomprises an amino acid L, V, or K at position 11 according to Kabatnumbering.
 18. The method of claim 13, wherein the modifiedimmunoglobulin single variable domain comprises an amino acid A or P atposition 14 according to Kabat numbering.
 19. The method of claim 13,wherein the modified immunoglobulin single variable domain comprises anamino acid A or P at position 41 according to Kabat numbering.
 20. Themethod of claim 13, wherein the modified immunoglobulin single variabledomain comprises an amino acid T, V, or L at position 89 according toKabat numbering.
 21. The method of claim 13, wherein the immunoglobulinsingle variable domain is comprised in a protein, polypeptide or otherconstruct.
 22. A method of modifying an immunoglobulin single variabledomain (VH or VHH) to have reduced binding to its C-terminal end bypre-existing antibodies found in human blood or serum, the methodcomprising mutating the amino acid sequence of the immunoglobulin singlevariable domain at position 11, at position 89, or at positions 11 and89 to generate a modified immunoglobulin single variable domaincomprising the amino acid V at position 11 and the amino acid L atposition 89, wherein the positions are numbered according to Kabatnumbering.
 23. The method of claim 22, further comprising extending theC-terminal end of said immunoglobulin single variable domain by 1 to 5amino acid residues.